Abstract
Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumor-derived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the “off switch” of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the “on switch” of the hypothetically oncogenic reduction activity of IDH1 by this mutation.
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Abbreviations
- ICT:
-
(isocitrate)
- αKG:
-
(α-ketoglutarate)
- 2HG:
-
(2-hydroxyglutarate)
- IDH:
-
(isocitrate dehydrogenase)
- NADP-IDH:
-
(NADP-dependent IDH)
- EcIDH:
-
(E. coli NADP-IDH)
- IDH1:
-
(human cytosolic NADP-IDH)
- PmIDH:
-
(porcine mitochondrial NADP-IDH)
- Idp1:
-
(S. cerevesiae mitochondrial NADP-IDH)
- GBM:
-
(glioblastoma multiforme)
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Acknowledgements
We thank the staff members at Photon Factory of Japan and Shanghai Synchrotron Radiation Facility (SSRF) of China for technical supports in diffraction data collection, and Sheng Li, Jian Wu, and Xianchi Dong of our group for technical assistance and helpful discussion. This work was supported by Grants from the Ministry of Science and Technology of China (2006AA02A313 and 2007CB914302), the National Natural Science Foundation of China (30730028), and the Chinese Academy of Sciences (KSCX2-YW-R-107 and SIBS2008002).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Table S1
The undetected regions of seg1 and seg2 in the R132H mutant complexes (PDF 7 kb)
Supplementary information, Table S2
Hydrogen-bonding interactions between the two flexible regions in the wild-type IDH1 complexes (PDF 17 kb)
Supplementary information, Figure S1
Mechanisms of “on switch” of the αKG reductase activity of IDH1 by the tumor-associated mutation R132H. (PDF 93 kb)
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Yang, B., Zhong, C., Peng, Y. et al. Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H. Cell Res 20, 1188–1200 (2010). https://doi.org/10.1038/cr.2010.145
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DOI: https://doi.org/10.1038/cr.2010.145
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