Fig. 6: Tyr35-Tyr35 interactions between two (NIA)2-bound ISCU2 are crucial for triggering [2Fe-2S] cluster formation by ISCU2 dimerization.
From: N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization
a Di-tyrosine formation by UV-crosslinking. Chemically reconstituted ISCU2 but not ISCU2-Y35D (lacking N-terminal Tyr35, but having two other Tyr) show a fluorescence increase (arrow) at λEx 320 nm and λEm 380–460 nm upon UV irradiation characteristic for di-tyrosine formation. The spectra recorded before and after crosslinking are set to the values at λEm 380 nm. The inset shows an immunoblot of a reducing SDS-PAGE from the UV-treated samples stained for the HIS-tag of the ISCU2 proteins. The band marked by the arrow represents the di-tyrosine-linked ISCU2 dimer. The experiment was repeated twice with similar outcome. b Only (NIA)2-bound ISCU2 and not free ISCU2 gives rise to holo-ISCU2 dimer formation. For experimental setup see Supplementary Fig. 10a. Representative aSEC profiles are shown for absorption at 280 nm (protein), 320 nm (Fe/S cluster) and 420 nm (Fe/S cluster and PLP). c Fractions from peaks 1 to 3 of part b containing (NIA)2, dimeric ISCU2-HIS and monomeric ISCU2, respectively, were analyzed by SDS-PAGE and subsequent anti-HIS tag immunostaining or Coomassie staining. The reaction mixture prior to Cys addition (sample 0) served as a control (cf. Supplementary Fig. 10a). The experiment was repeated twice with similar outcome. Source Data are provided as a Source Data file.
