Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1

Abstract

[4Fe-4S]²⁺ cluster assembly in human cytosol requires both a [2Fe-2S] cluster chaperone being able to donate two [2Fe-2S]²⁺ clusters and an electron donor providing two electrons to reductively couple the two [2Fe-2S]²⁺ clusters into a [4Fe-4S]²⁺ cluster. The mechanism through which the cytosolic [4Fe-4S]²⁺ cluster assembly works is still not defined. Here, we show that a hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S]⁺ ₂ -anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S]²⁺ ₂ -GLRX3₂ orchestrates the assembly of a [4Fe-4S]²⁺ cluster on the N-terminal cluster binding site of the cytosolic protein NUBP1. We demonstrate that the hetero-tetrameric complex is able to synergically provide two [2Fe-2S]²⁺ clusters from GLRX3 and two electrons from anamorsin for the assembly of the [4Fe-4S]²⁺ cluster on the N-terminal cluster binding site of NUBP1. We also showed that only one of the two [2Fe-2S] clusters bound to anamorsin, that is, that bound to the CX₈ CX₂ CXC motif, provides the electrons required to form the [4Fe-4S]²⁺ cluster. Our study contributes to the molecular understanding of the mechanism of [4Fe-4S] protein biogenesis in the cytosol.

Keywords: GLRX3; NUBP1; anamorsin; electron transfer; iron-sulfur protein biogenesis.

FIGURE 1

[2Fe‐2S]²⁺ ₂‐GLRX3₂/[2Fe‐2S]⁺ ₂‐anamorsin mixture promotes the assembly of a [4Fe‐4S]²⁺ cluster at the N‐terminal site of His₆‐tagged NUBP1. (A) UV–vis spectra of His₆‐tagged NUBP1 before (blue line) and after (black line) the incubation and isolation from [2Fe‐2S]²⁺ ₂‐GLRX3₂ and [2Fe‐2S]⁺ ₂‐anamorsin; (B) paramagnetic 1D ¹H NMR spectrum of His₆‐tagged NUBP1 after incubation and isolation from [2Fe‐2S]²⁺ ₂‐GLRX3₂ and [2Fe‐2S]⁺ ₂‐anamorsin.

FIGURE 2

[2Fe‐2S]⁺ ₂‐anamorsin provides electrons and [2Fe‐2S]²⁺ ₂‐GLRX3₂ provides the [2Fe‐2S]²⁺ clusters for the assembly of a [4Fe‐4S]²⁺ cluster at the N‐terminal site of His₆‐tagged NUBP1. UV–vis spectra of [2Fe‐2S]²⁺ ₂‐GLRX3₂/[2Fe‐2S]⁺ ₂‐anamorsin mixture before (cyan line) and after (black line) the incubation and separation from His₆‐tagged NUBP1. The spectra of isolated [2Fe‐2S]²⁺ ₂‐GLRX3₂ (green line), [2Fe‐2S]⁺ ₂‐anamorsin (blue line) and [2Fe‐2S]²⁺ ₂‐anamorsin (red line) are reported for reference.

FIGURE 3

The M1‐bound [2Fe‐2S]⁺ cluster of anamorsin provides electrons for the assembly of the [4Fe‐4S]²⁺ cluster at the N‐terminal site of NUBP1. (A) UV–vis spectra of His₆‐tagged NUBP1 before (black line) and after the incubation and isolation from [2Fe‐2S]²⁺ ₂‐GLRX3₂ and M1‐ (red line) and M2‐anamorsin (blue line); (B) paramagnetic 1D ¹H NMR spectra of His₆‐tagged NUBP1 after incubation and isolation from [2Fe‐2S]²⁺ ₂‐GLRX3₂ and [a] M1‐ and [b] M2‐anamorsin (5 times magnification).

FIGURE 4

The M2‐bound [2Fe‐2S]⁺ cluster of anamorsin does not transfer electrons for the assembly of a [4Fe‐4S] cluster on NUBP1. (A) CW X‐band EPR spectra at 10 K, 1 mW of: cluster‐oxidized [2Fe‐2S]²⁺ ₂‐WT‐anamorsin (gray line); dithionite reduced [2Fe‐2S]⁺ ₂‐WT‐anamorsin (green line); a mixture of [2Fe‐2S]²⁺ ₂‐GLRX3₂ and dithionite reduced [2Fe‐2S]⁺ ₂‐WT‐anamorsin (magenta line); a mixture of [2Fe‐2S]²⁺ ₂‐GLRX3₂, dithionite reduced [2Fe‐2S]⁺ ₂‐WT‐anamorsin and NUBP1 (black line); dithionite reduced [2Fe‐2S]⁺‐M1‐anamorsin (blue line); dithionite reduced [2Fe‐2S]⁺‐M2‐anamorsin (red line). g values for reduced M1‐ and M2‐bound [2Fe‐2S]⁺ clusters are indicated in blue and red, respectively. The black, dotted arrows indicate the decrease in intensity of the EPR signal of the reduced M1‐bound [2Fe‐2S] cluster of [2Fe‐2S]⁺ ₂‐WT‐anamorsin upon addition of His₆‐tagged NUBP1 to the mixture of [2Fe‐2S]²⁺ ₂‐GLRX3₂ and dithionite reduced [2Fe‐2S]⁺ ₂‐WT‐anamorsin. (B) From top to bottom: 1D ¹H paramagnetic NMR spectra of a mixture of [2Fe‐2S]²⁺ ₂‐GLRX3₂, dithionite reduced [2Fe‐2S]⁺ ₂‐WT‐anamorsin and apo NUBP1; isolated dithionite reduced [2Fe‐2S]⁺‐M2‐anamorsin; isolated [2Fe‐2S]²⁺ ₂‐GLRX3₂; isolated cluster‐oxidized [2Fe‐2S]²⁺ ₂‐WT‐anamorsin. NMR spectra were acquired at 400 MHz, 298 K.

FIGURE 5

[2Fe‐2S]₂‐GLRX3₂ and [2Fe‐2S]₂‐anamorsin form a hetero‐tetrameric complex that drives the assembly of the [4Fe‐4S]²⁺ cluster at the N‐terminal site of NUBP1. (A) Analytical SEC of [2Fe‐2S]₂‐GLRX3₂/[2Fe‐2S]₂‐WT‐anamorsin mixtures composed as indicated in the figure. Vertical, dotted lines indicate the peaks corresponding to the main species: cyan = apo GLRX3; green = [2Fe‐2S]₂‐GLRX3₂; blue = [2Fe‐2S]²⁺ ₂‐anamorsin; red = a 1:2 [2Fe‐2S]₂‐GLRX3₂:[2Fe‐2S]₂‐anamorsin complex. (B) SDS‐PAGE analysis of the protein content of the SEC peak corresponding to the hetero‐tetrameric 1:2 [2Fe‐2S]₂‐GLRX3₂:[2Fe‐2S]₂‐anamorsin complex in panel A. M = marker; lane 1 = isolated [2Fe‐2S]₂‐anamorsin; lane 2 = isolated [2Fe‐2S]₂‐GLRX3₂; lane 3 = main peak of the top line in panel A, corresponding to the GLRX3/anamorsin hetero‐tetrameric complex. (C) Analytical SEC of the products of the cluster transfer/assembly reaction before and after separation by Ni²⁺‐affinity chromatography, of apo His₆‐tagged NUBP1 before incubation and of the 1:1 apo GLRX3/[2Fe‐2S]₂‐anamorsin heterodimeric complex. Vertical, dotted lines indicate the peaks corresponding to the main species: purple = apo GLRX3:[2Fe‐2S]₂‐anamorsin 1:1 complex; magenta = His₆‐tagged NUBP1 before and after the reaction.

SCHEME 1

Proposed model for the [4Fe‐4S]²⁺ cluster assembly process on the N‐terminal site of NUBP1. [2Fe‐2S]²⁺ ₂‐GLRX3₂ and [2Fe‐2S]⁺ ₂‐anamorsin interact forming a 1:2 [2Fe‐2S]²⁺ ₂‐GLRX3₂:[2Fe‐2S]⁺ ₂‐anamorsin hetero‐tetrameric complex. The complex formation is driven by specific protein–protein recognition between the two N‐terminal domains of GLRX3 and anamorsin, according to what previously reported (Banci, Ciofi‐Baffoni, et al., 2015). The hetero‐tetrameric complex transiently interact with NUBP1 and transfers the two [2Fe‐2S]²⁺ clusters of GLRX3 and two electrons from the reduced M1‐bound [2Fe‐2S]⁺ clusters of anamorsin to assemble a [4Fe‐4S]²⁺ cluster on the N‐terminal site of NUBP1. As a result of both cluster and electron transfer, the hetero‐tetrameric complex is converted into a 1:1 apo GLRX3:[2Fe‐2S]₂‐anamorsin complex. Fe–S clusters are represented as yellow spheres (sulfur atoms) and red spheres (Fe³⁺) or green spheres (Fe²⁺). GS stands for the cluster‐bound form of glutathione (GSH), which interacts with GLRX3 fulfilling the [2Fe‐2S] cluster first coordination sphere in [2Fe‐2S]²⁺ ₂‐GLRX3₂. After the cluster transfer event, GSH still interacts with GLRX3.