The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis

Abstract

Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro. Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named Mrp_ORP associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length Mrp_ORP, as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the Mrp_ORP can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.

Figure 1

Unrooted Maximum likelihood tree of the Mrp/Nbp35 ATP-binding protein family (IPR019591, 16154 sequences, 114 amino acid positions conserved for the analysis after trimming). The tree is displayed as a cladogramme. Branch colors correspond to taxonomic groups: pink = Bacteria, blue = Eucarya, green = Archaea. DVU2109, DVU1847, and DVU2330 from Desulfovibrio vulgaris strain Hildenborough ATCC 29579, Dde3202 from Desulfovibrio alaskensis strain G20, Mrp from Escherichia coli strain K12, ApbC from Salmonella typhimurium strain LT2 SGSC1412 ATCC 700720, Cfd1 and Nbp35 from Saccharomyces cerevisiae strain ATCC 204508 S288c are indicated by red triangles. The 99 sequences associating a P-loop NTPase domain and a Di-Nase domain are indicated by purple triangles. Because of the large number of sequences contained in the tree, some triangles may overlap.

Figure 2

Protein sequences alignment of bacterial Mrp/ApbC and eukaryotic Nbp35 homologs. Bacterial sequences are from Dde (DDE_3202, UniProt accession no. Q30WF0), DvH (DVU2109, UniProt accession no. Q72A88), E. coli (Mrp, UniProt accession no. P0AF08) and S. enterica (ApbC, UniProt accession no. Q8ZNN5). The eukaryotic homologs are the paralogs from S. cerevisiae (Nbp35, UniProt accession no. P52920 and Cfd1; UniProt accession no. P40558). Conserved amino acids are shown on a black background. Conserved cysteine residues are indicated with an asterisk black or a blue cross above the residue. Mutated cysteine in alanine residues are indicated with a red asterisk above the residue. The blue frame corresponds to the P-loop NTPase end the orange to the Di-Nase domain. The sequence alignment was built using the clustal O program https://www.ebi.ac.uk/Tools/msa/clustalo/ (version 1.2.1).

Figure 3

UV-visible absorption spectra of oxidized and reduced wild-type full, P-loop NTPase domain and mutant Mrp_{ORP C215A-C218A} proteins. UV-visible absorption spectra of aerobically isolated wild-type Mrp_ORP (dotted line)_, enzymatically reconstituted the Mrp_ORP protein before (solid bold line) and after reduction with addition of sodium dithionite (solid grey line) and enzymatically reconstituted Mrp_{ORPC215A,C218A} protein before (dashed bold line) and after reduction with addition of sodium dithionite (dashed grey line). Inset: UV-visible absorption spectra of the enzymatically Fe-S reconstituted P-loop NTPase domain before (solid line) and after reduction with addition of sodium dithionite (dashed line).

Figure 4

The Di-Nase of Mrp_ORP binds a 3Fe4S cluster. (a) UV-visible absorption spectra of the anaerobically purified Di-Nase domain of Mrp_ORP protein isolated from DvH before (solid line) and after reduction by addition of sodium dithionite (dashed line). (b) EPR spectra of 80 μM (i) as prepared and (ii) dithionite reduced Mrp_ORP in 50 mM Tris-HCl 8.1, 150 mM NaCl, 1 mM DTT. Instrument settings: microwave frequency, 9.66 GHz; modulation amplitude, 5 G; microwave power, 6 mW; gain, 1 × 10⁵ temperature, 20 K. An impurity signal, designated by * arises from a trace species present in the cavity baseline. (c) Cyclic voltammogram of 121 µM Di-Nase domain of Mrp_ORP on a PGE electrode coated with 1.5 µL of polymyxin B sulphate (2 mM), in 100 mM Tris-HCl pH 8.0, 500 mM NaCl, 3 mM DTT and 2.5 mM desthiobiotin at a scan rate of 20 mV.s⁻¹. Dash line represents the blank voltammogram and solid line the voltammogram of the protein film. The arrow indicates the direction of the scan.

Figure 5

Reconstituted Mrp_ORP is able to transfer its [Fe-S] clusters to Apo-AcnB. The graph shows the correlation between aconitase activity and pre-incubation time between Rec-Mrp_ORP (16 µM) and Apo-AcnB (2 µM). 100% of activity corresponds to the specific activity of AcnB recorded with reconstituted AcnB in the same condition. Data points show the average of two experiments. (b) Correlation between the increasing level of Rec-Mrp_ORP and aconitase activity (full square). AcnB activation assays contained 2 µM of apo-AcnB protein and 0 to 40 µM of Rec-Mrp_ORP. Apo-AcnB and reconstituted Mrp_ORP proteins were incubated together during 30 minutes before recording aconitase activity. Aconitase activity was measured at 30 °C during 1 h at 340 nm under anaerobiosis. (c) Aconitase activities relative to the full-length Rec-Mrp_ORP determined with 15 µM of Fe²⁺ and S²⁻Rec-, 15 µM of P-loop NTPase domain, 20 µM of Di-Nase domain and 4 µM of Rec_AcnB. Data are represented as the average of two independent experiments with standard deviations shown as error bars.

Figure 6

Mrp_ORP transfers its [Fe-S] cluster to ORP protein_S. Apo-Orp3-Orp4-Orp8 proteins (dashed line) were mixed with Holo-rp_ORP during 90 minutes in anaerobic condition. After separation on a Ni-NTA column, the UV-visible absorption spectrum of the eluate fraction containing Orp3 was recorded (solid line). Inset: UV-visible spectrum of holo- Orp3-Orp4-Orp8 purified in anaerobiosis condition from DvH.

Figure 7

Polar and dynamic subcellular localization of Mrp_ORP-GFP. Subcellular localization of Mrp_ORP-GFP. (a) Subcellular localization of full length Mrp_ORP-GFP. The first column represents the phase-contrast images (a), the second represents the nucleoid localization (DAPI) (b), the third represents the Mrp_ORP-GFP fluorescence (c) and the fourth represents an overlay of both fluorescence signals (d). Scale bar = 1 µm. (b) Subcellular localization of the P-loop NTPase-GFP fusion protein. The first column represents the phase-contrast images (a), the second represents the nucleoid localization (DAPI) (b), the third represents the P-loop NTPase -GFP fluorescence (c), the fourth represents the membrane localization (FM4–64) (d) and the sixth represents an overlay of all flurescence signals (e). Scale bar = 1 µm. (c) Subcellular localization of the Di-Nase-GFP fusion protein. The first column represents the phase-contrast images (a) and the second represents the Mrp_ORP-GFP fluorescence (b). Scale bar = 2 µm. (d) Statistical analysis of the the number of foci per cell located at the cellular pole in Mrp_ORP during the initiation step of the cell cycle of DvH was performed using 362 cells from 3 independent experiments. (e) Statistical analysis of the number of foci per cell located at the cellular pole of the P-Loop NTPase domain during the initiation step of the cell cycle of DvH was performed using 1232 cells from 3 independent experiments.