title: A point mutation in the [2Fe-2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties.
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 1.65 Å 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 1.58 Å 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 300 mV 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.
authors.affiliation: The Alexander Silberman Life Science Institute and the Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904, Israel.
authors.forename: Sagi
authors.initials: S
authors.lastname: Tamir
authors.affiliation: The Alexander Silberman Life Science Institute and the Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904, Israel.
authors.forename: Yael
authors.initials: Y
authors.lastname: Eisenberg-Domovich
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Conlan
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Stofleth
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Colin H
authors.initials: CH
authors.lastname: Lipper
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Paddock
authors.affiliation: Department of Biology, University of North Texas, Denton, TX 76203, USA.
authors.forename: Ron
authors.initials: R
authors.lastname: Mittler
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
authors.affiliation: The Alexander Silberman Life Science Institute and the Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904, Israel.
authors.forename: Oded
authors.initials: O
authors.lastname: Livnah
authors.affiliation: The Alexander Silberman Life Science Institute and the Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904, Israel.
authors.forename: Rachel
authors.initials: R
authors.lastname: Nechushtai
mesh_terms: D018360:Crystallography, X-Ray; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D063347:Native Polyacrylamide Gel Electrophoresis; D017354:Point Mutation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007506:Iron-Sulfur Proteins; D008024:Ligands
keywords: 2Fe–2S cluster transfer and cluster stability; WFS2; autophagy; breast cancer; longevity; redox potential