title: Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.
abstract: Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
authors.forename: Surinder M
authors.initials: SM
authors.lastname: Singh
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
authors.forename: Swati
authors.initials: S
authors.lastname: Bandi
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
authors.forename: Dinen D
authors.initials: DD
authors.lastname: Shah
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, United States of America.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Armstrong
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America; Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
authors.forename: Krishna M G
authors.initials: KM
authors.lastname: Mallela
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D055592:Biophysical Phenomena; D002311:Cardiomyopathy, Dilated / Q000235:genetics; D016189:Dystrophin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding
chemical_list: D000199:Actins; D000596:Amino Acids; D016189:Dystrophin; D050505:Mutant Proteins