Local Destabilization of the Metal-Binding Region in Human Copper−Zinc Superoxide Dismutase by Remote Mutations Is a Possible Determinant for Progression of ALS
Abstract
More than 100 distinct mutations in the gene CuZnSOD encoding human copper–zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where ^(15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer β-strands I and VI of the β-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817–8829) of amide hydrogen exchange at pH 7 and 25 °C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local β-strands.
Additional Information
© 2014 American Chemical Society. Received: May 19, 2014. Revised: December 14, 2014. Published: December 15, 2014. The study was supported by grants from the Swedish Research Council to B.-H.J. (Dnr 2006-4253) and P.L. (Dnr 621-2012- 5136). We thank the Swedish NMR center in Gothenburg for measurement time at the 800 MHz spectrometer. J.H. gratefully acknowledges Forum Scientium for financial support, the Swedish Research Council (Vetenskapsrådet), and the European Molecular Biology Organization (EMBO, ALTF 276-2010) for postdoc fellowships.Attached Files
Supplemental Material - bi500606j_si_001.pdf
Files
Name | Size | Download all |
---|---|---|
md5:a95e63cc093da1734fd54927ef3f5b97
|
466.8 kB | Preview Download |
Additional details
- Eprint ID
- 55658
- Resolver ID
- CaltechAUTHORS:20150310-080955818
- Forum Scientium
- Swedish Research Council (Vetenskapsrådet)
- ALTF 276-2010
- European Molecular Biology Organization (EMBO)
- Dnr 2006-4253
- Swedish Research Council
- Dnr 621-2012-5136
- Swedish Research Council
- Created
-
2015-03-10Created from EPrint's datestamp field
- Updated
-
2021-11-10Created from EPrint's last_modified field