Fig 1.
Structures and UV-Vis absorption spectra of NEET proteins and Anamorsin.
A. (Top) Crystal structures of mNT (PDB code: 2QH7,), NAF-1 (PDB code: 3FNV); (Middle) NEET 2Fe-2S cluster with 3-Cys:1His coordination; (Bottom) Absorption spectra of 25 μM mNT and NAF-1. B. (Top) Crystal structure of the N-terminal domain of Anamorsin (PDB code: 2YUI) with an added schematic of the unstructured 2Fe-2S cluster binding domain; (Middle) Representative Anamorsin 2Fe-2S cluster with 4-Cys coordination (from ferredoxin, PDB code: 1RFK); (Bottom) Absorption spectrum of 43 μM Anamorsin isolated from E. coli.
Fig 2.
NEET proteins transfer their 2Fe-2S cluster to apo-Anamorsin.
The 2F-2S cluster transfer reaction was monitored by UV-Vis absorption spectroscopy. The progress of the transfer was plotted versus time. Cluster transfer from NAF-1 (A) and mNT (B) to apo-Anamorsin occurs only when the 2Fe-2S cluster is oxidized and not when reduced with 10 mM sodium dithionite. Replacement of the coordinating His residue with Cys (H114C for NAF-1 and H87C for mNT) inhibits but does not abolish transfer. All traces shown were obtained with 25 μM NAF-1 or mNT (50 μM 2Fe-2S clusters) and 50 μM apo-Anamorsin in 50 mM bis tris, 100 mM NaCl, 2.5 mM DTT, pH 7.0 at 37°C.
Fig 3.
NEET transfer of 2Fe-2S clusters to apo-Anamorsin is second order.
NAF-1 (A) and mNT (B) transfer to apo-Anamorsin was monitored by UV-Vis absorption spectroscopy for a series of NEET concentrations. For each NEET concentration the ratio 1 NEET dimer per 2 apo-anamorisn was maintained. The rate constant, kobs, is determined from the fit of the data to an exponential rise and is plotted versus concentration for NAF-1 (C) or mNT (D). The slope of the best line fit was used to determine apparent second order rate constants (k2) for NAF-1 and mNT, which are 600 ± 90 M-1 min-1 and 460 ± 60 M-1 min-1 respectively.
Table 1.
Comparision of NEET 2Fe-2S cluster transfer rates to apo-Anamorsin with other known cluster transfer proteins.
Fig 4.
Biolayer interferiometry shows a direct protein-protein interaction of NAF-1 and mNT with apo-Anamorsin.
Sensorgrams for the binding of holo-NAF-1 (A) and holo-mNT (B) to biotinylated apo-Anamorsin immobilized to streptavidin-coated biosensors are shown. The association was followed for 900 seconds (rising signal) followed by 1800 seconds of dissociation (decaying signal). The data was fit to a one-to-one model (black curves). On- and off-rates were determined from the fits for each NEET-Anamorsin concentration (shown in Table 2).
Table 2.
NEET-apo-Anamorsin association and dissociation rates determined by biolayer interferometry.
Fig 5.
NEET proteins can transfer their clusters to either of Anamorsin 2Fe-2S cluster-binding sites.
Anamorsin mutants containing a single cluster-binding site are shown; the other cluster site was disrupted by replacing all of the Cys residues with Ser. (A) Schematics of Anamorsin-C1 (green) and Anamorsin-C2 (magenta) are shown. 2Fe-2S cluster transfer from NAF-1 (B) or mNT (C) to each single cluster binding apo-Anamorsin mutant was monitored by UV-Vis absorption spectroscopy. Traces were obtained with 25 μM NAF-1 or mNT (50 μM 2Fe-2S clusters) and 50 μM apo-Anamorsin.
Fig 6.
NEET homodimers can transfer their two 2Fe-2S clusters to Anamorsin.
(A) 2Fe-2S transfer from 50 μM dimeric NAF-1 to 50 μM apo-Anamorsin (two 2Fe-2S clusters per Anamorsin) (shown in red) is compared to 25 μM NAF-1 to 50 μM apo-Anamorsin (one 2Fe-2S cluster per Anamorsin) (shown in blue). Transfer is near complete for the one 2Fe-2S cluster per Anamorsin sample and is approximately 75% complete for the two 2Fe-2S cluster per Anamorsin sample. The latter result shows that at least half of the Anamorsin is capable of receiving two 2Fe-2S clusters from a single NAF-1 homodimer. (B) Anamorsin following a transfer reaction with NAF-1 was purified and analyzed by ESI-MS (shown in red). Mass spectra of apo-Anamorsin (solid black line) is compared to the major peak for post-transfer Anamorsin (solid red line) which shows an increase in the mass corresponding to the incorporation of two 2Fe-2S clusters. There is also a minor peak at 35856 Da that likely corresponds to Anamorsin with a single 2Fe-2S cluster and an iron ion bound that may be a remnant of a cluster that degraded during the sample preparation process. E. coli-purified Anamorsin is shown for comparison (dotted line) which shows Anamorsin containing a single 2Fe-2S cluster.
Fig 7.
NEET proteins provide a link between the ISC and CIA pathways.
mNT on the OMM and NAF-1 on the MAM receive 2Fe-2S clusters produced inside the mitochondria by the ISC system. Both mNT and NAF-1 transfer these 2Fe-2S clusters to Anamorsin. Anamorsin receives electrons from the diflavin reductase NDOR1 and supplies them to the CIA system as an early step necessary for the production of 4Fe-4S clusters. This step requires the holo form of Anamorsin. Both mNT and NAF-1 can provide parallel routes linking CIA to ISC. CIA produced clusters are targeted to proteins in the cytosol and the nucleus and are important for cell metabolism, maintenance and proliferation.