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.

Keywords: 2Fe–2S cluster transfer and cluster stability; WFS2; autophagy; breast cancer; longevity; redox potential.

Figure 1

The 2F _o − F _c electron-density map at 1.5σ depicting the [2Fe–2S] cluster and its interactions with NAF-1 for (a) NAF-1 and (b) NAF-1 H114C.

Figure 2

The crystal structure of NAF-1 consists of an interlaced dimer (left; shown in blue and cyan). The structure of NAF-1 H114C exhibits a highly similar topology (center), as can also be observed in the superposition (right).

Figure 3

The stability of the [2Fe–2S] cluster of NAF-1 H114C is greatly increased. The cluster stabilities of NAF-1 (blue), NAF-1 H114C (red) and Fd (green) were monitored over time by optical spectroscopy at 458 nm, which is attributed to cluster absorbance at 37°C. The absorbance decay corresponds to loss of the [2Fe–2S] cluster. The cluster is much more stable in NAF-1 H114C compared with native NAF-1 protein and its stability is almost equal to that in Fd.

Figure 4

Substitution of a single residue, His114, by Cys in NAF-1 alters the redox potential of the [2Fe–2S] cluster (E _m) of NAF-1. E _m of the NAF-1 H114C [2Fe–2S] cluster is shifted by >−300 mV compared with native NAF-1. Native NAF-1 (black) has a redox potential of approximately 24 mV (±5 mV). The point mutant H114C (blue) has a substantially shifted redox potential of −280 mV (±10 mV).

Figure 5

The NAF-1 [2Fe–2S] cluster-transfer function is abrogated in NAF-1 H114C. NAF-1 was incubated at 37°C for 20 min with β-mercaptoethanol pre-reduced apo-Fd and run on a native gel. In the upper native gels in each panel the red-colored bands are indicative of the [2Fe–2S] cluster in the two proteins; the upper red band represents NAF-1 (labeled NAF-1 on the right side of the gel) and the lower red band represents holo-Fd (labeled Fd). (a) Cluster transfer from NAF-1 to the pre-reduced apo-Fd in the absence and presence of MgCl₂. The native (WT) NAF-1 (lane 1) indicated by the red-colored upper band decreases in intensity upon incubation with pre-reduced apo-Fd, with a concomitant increase in the intensity of the lower red band owing to the formation of holo-Fd (lane 2). In the presence of MgCl₂, the cluster transfer from NAF-1 to apo-Fd is slightly greater (lane 3). NAF-1 H114C completely lost the cluster-transfer function in the absence (lane 4) or presence (lane 5) of MgCl₂. Lane 6 contains holo-Fd for reference. The lower gel was stained with Coomassie Blue to confirm that the protein levels were the same in all experiments. (b) Cluster transfer in the presence of glycerol. The native NAF-1 (WT; lane 1), when incubated with pre-reduced apo-Fd, transfers its cluster to the latter to form holo-Fd (lower red band, lane 2). The presence of glycerol prevents cluster transfer from NAF-1 to pre-reduced apo-Fd (lane 3). NAF-1 H114C does not function as a cluster-donor protein in the absence (lane 4) or the presence of glycerol (lane 5). Lane 6 contains the control holo-Fd.