TY - JOUR
T1 - Disease-causing missense mutations in actin binding domain 1 of dystrophin induce thermodynamic instability and protein aggregation
AU - Henderson, Davin M.
AU - Lee, Ann
AU - Ervasti, James M.
PY - 2010/5/25
Y1 - 2010/5/25
N2 - Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) most commonly through loss of protein expression. In a small subpopulation of patients, missense mutations can cause DMD, Becker muscular dystrophy, or X-linked cardiomyopathy. Nearly one-half of disease-causing missense mutations are located in actin-binding domain 1 (ABD1) of dystrophin. To test the hypothesis that ABD1 missense mutations cause disease by impairing actin-binding activity, we engineered the K18N, L54R, D165V, A168D, L172H, and Y231N mutations into the full-length dystrophin cDNA and characterized the biochemical properties of each mutant protein. The K18N and L54R mutations are associated with the most severe diseases in humans and each caused a small but significant 4-fold decrease in actin-binding affinity, while the affinities of the other four mutant proteins were not significantly different from WT dystrophin. More interestingly, WT dystrophin was observed to unfold in a single-step, highly cooperative manner. In contrast, all six mutant proteins were significantly more prone to thermal denaturation and aggregation. Our results suggest that missense mutations in ABD1 may all cause loss of dystrophin function via protein instability and aggregation rather than through loss of ligand binding function. However, more severe disease progressions may be due to the combinatorial effects of some mutations on both protein aggregation and impaired actin-binding activity.
AB - Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) most commonly through loss of protein expression. In a small subpopulation of patients, missense mutations can cause DMD, Becker muscular dystrophy, or X-linked cardiomyopathy. Nearly one-half of disease-causing missense mutations are located in actin-binding domain 1 (ABD1) of dystrophin. To test the hypothesis that ABD1 missense mutations cause disease by impairing actin-binding activity, we engineered the K18N, L54R, D165V, A168D, L172H, and Y231N mutations into the full-length dystrophin cDNA and characterized the biochemical properties of each mutant protein. The K18N and L54R mutations are associated with the most severe diseases in humans and each caused a small but significant 4-fold decrease in actin-binding affinity, while the affinities of the other four mutant proteins were not significantly different from WT dystrophin. More interestingly, WT dystrophin was observed to unfold in a single-step, highly cooperative manner. In contrast, all six mutant proteins were significantly more prone to thermal denaturation and aggregation. Our results suggest that missense mutations in ABD1 may all cause loss of dystrophin function via protein instability and aggregation rather than through loss of ligand binding function. However, more severe disease progressions may be due to the combinatorial effects of some mutations on both protein aggregation and impaired actin-binding activity.
KW - Calponin homology domain
KW - Muscular dystrophy
KW - Thermal stability
UR - http://www.scopus.com/inward/record.url?scp=77953108170&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77953108170&partnerID=8YFLogxK
U2 - 10.1073/pnas.1001517107
DO - 10.1073/pnas.1001517107
M3 - Article
C2 - 20457930
AN - SCOPUS:77953108170
SN - 0027-8424
VL - 107
SP - 9632
EP - 9637
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 21
ER -