Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration.
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We would like to thank Dr. Mary E. Porter for providing the pET-30a(+) expression vector. We very much appreciate Dr. James M. Ervasti lab members Jackie L. McCourt and Dr. Joseph J. Belanto for providing an F-actin co-sedimentation protocol and assistance with circular dichroism data analyses. Further we thank Dr. David D. Thomas lab members Benjamin P. Binder for instruction in PyMOL and Discovery Studio Visualizer and Dr. Bengt Svensson for assistance in structural homology modeling. We very much appreciate Dr. Thomas Hays lab members Dr. Amanda L. Neisch, Dr. Min-gang Li and Akshaya K. Gupta for intellectual input and technical assistance. This work was supported by a post-doctoral fellowship to A.W. Avery from the National Ataxia Foundation and Bob Allison Ataxia Research Center, and by a grant from the National Institutes of Health (RO1GM44757) to T.S. Hays.