The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4

Abstract

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The E. coli genome encodes members of both of these proteins families, namely the monothiol glutaredoxin Grx4, and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimers as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

Schizosaccharomyces pombe Grx4 regulates the transcriptional repressor Php4 via [2Fe–2S] cluster binding

“…27 Previously published spectra of [2Fe-2S] Grxs purified under strictly anaerobic conditions typically have two distinct peaks at ~320 and ~410 nm, with a characteristic shoulder at ~445 nm. 11,13,28,29 To confirm that S. pombe Grx4 binds a bona fide [2Fe-2S] 2+ cluster similar to previously characterized Grxs, we overexpressed and purified recombinant S. pombe Grx4 from E. coli . The purified protein had a reddish-brown color, and was investigated by spectroscopic and biochemical methods.…”

“…This signal is similar to other [2Fe-2S]-bridged CGFS Grxs, which are also known to be reductively labile, resulting in a low number of spins per cluster. 13,28,29 [2Fe-2S] + Php4-Grx4 also gives a rhombic S = ½ EPR signal, g 1,2,3 = 2.00, 1.96, and 1.92 ( g av = 1.96), accounting for ~20% of the cluster content (Fig. 3B, bottom).…”

“…15,25,28,29,33,34 Furthermore, [2Fe-2S]-bridged Grx homodimers and [2Fe-2S]-bridged Grx-BolA heterocomplexes can transfer Fe-S clusters to specific apo acceptor proteins. 11,15,18,23,27,33,35,36 Of note, the [2Fe-2S] Grx3-Fra2 complex in S. cerevisiae specifically transfers its cluster to the transcription factor Aft2 when these proteins are mixed.…”

Characterization of Glutaredoxin Fe–S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy

“…In addition, the [2Fe-2S] concentration can be estimated with UV-visible absorption spectroscopy from the dominant band in the 390–430 nm range. The extinction coefficient for this peak is typically in the range of 8–11 mM −1 cm −1 per [2Fe-2S] cluster for Grx homodimers and 5–9 mM −1 cm −1 per [2Fe-2S] cluster for Grx heterocomplexes (Bandyopadhyay et al, 2008; Dlouhy et al, 2016; Li et al, 2011; Li et al, 2009; Li et al, 2012; Nasta et al, 2017; Roret et al, 2014; Zhang et al, 2013), similar to other [2Fe-2S] 2+ cluster proteins (Dailey, Finnegan, & Johnson, 1994). Fe-S cluster loading and protein purity can thus be assessed from the A 390 :A 280 ratio calculated from the UV-visible absorption spectrum.…”

“…Cellular processes involving CGFS Grxs often proceed as Fe-S cluster-dependent protein-protein interactions via a mechanism that employs structural rearrangements and ligand exchange. Two primary types of Grx Fe-S cluster-dependent interactions have been identified thus far in which Grxs interact with one or two binding partners to form (1) a heterocomplex with the Fe-S cluster ligated at the interface of the Grx domain(s) and the interacting partner protein as illustrated by the Grx-BolA complexes (Banci, Camponeschi, Ciofi-Baffoni, & Muzzioli, 2015; Dlouhy et al, 2016; Li et al, 2009; Li, Mapolelo, Randeniya, Johnson, & Outten, 2012; Nasta, Giachetti, Ciofi-Baffoni, & Banci, 2017; Roret et al, 2014; Uzarska et al, 2016; Yeung et al, 2011), or (2) the formation of an entirely new Fe-S cluster coordinated protein complex obtained via complete and intact transfer of the Fe-S cluster from Grx to the interacting partner (Banci et al, 2014; Banci, Camponeschi, et al, 2015; Banci, Ciofi-Baffoni, et al, 2015; Bandyopadhyay et al, 2008; Fidai, Wachnowsky, & Cowan, 2016; Mapolelo et al, 2013; Poor et al, 2014; Qi & Cowan, 2011; Shakamuri, Zhang, & Johnson, 2012; Vranish, Das, & Barondeau, 2016; Vranish et al, 2015; Xia et al, 2015; Yeung et al, 2011; Zhang et al, 2013). …”