A point mutation in the [2Fe-2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties

Sagi Tamir, Yael Eisenberg-Domovich, Andrea R. Conlan, Jason T. Stofleth, Colin H. Lipper, Mark L. Paddock, Ron Mittler, Patricia A. Jennings, Oded Livnah*, Rachel Nechushtai

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

NAF-1 is an important [2Fe-2S] NEET protein associated with human health and disease. A mis-splicing mutation in NAF-1 results in Wolfram Syndrome type 2, a lethal childhood disease. Upregulation of NAF-1 is found in epithelial breast cancer cells, and suppression of NAF-1 expression by knockdown significantly suppresses tumor growth. Key to NAF-1 function is the NEET fold with its [2Fe-2S] cluster. In this work, the high-resolution structure of native NAF-1 was determined to 1.65Å resolution (R factor = 13.5%) together with that of a mutant in which the single His ligand of its [2Fe-2S] cluster, His114, was replaced by Cys. The NAF-1 H114C mutant structure was determined to 1.58Å resolution (R factor = 16.0%). All structural differences were localized to the cluster binding site. Compared with native NAF-1, the [2Fe-2S] clusters of the H114C mutant were found to (i) be 25-fold more stable, (ii) have a redox potential that is 300mV more negative and (iii) have their cluster donation/transfer function abolished. Because no global structural differences were found between the mutant and the native (wild-type) NAF-1 proteins, yet significant functional differences exist between them, the NAF-1 H114C mutant is an excellent tool to decipher the underlying biological importance of the [2Fe-2S] cluster of NAF-1 in vivo.

Original languageAmerican English
Pages (from-to)1572-1578
Number of pages7
JournalActa Crystallographica Section D: Biological Crystallography
Volume70
Issue number6
DOIs
StatePublished - Jun 2014

Keywords

  • 2Fe-2S cluster transfer and cluster stability
  • WFS2
  • autophagy
  • breast cancer
  • longevity
  • redox potential

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