Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins

Abstract

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or ⁵⁵Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.

Keywords: CIA machinery; NBP35; NUBP1-NUBP2; iron homeostasis; iron-sulfur protein.

Fig. 1.

Depletion of human CFD1 both impairs Fe-S cluster assembly on IRP1 and affects cellular iron regulation. HeLa cells were transfected at 3-d intervals with no siRNA (Mock), scrambled siRNAs (Scr), or a pool of CFD1-directed siRNAs (siCFD1). After each round of transfection cells were fractionated using 0.01% digitonin. (A) Total-cell lysates as well as cytosol and organelle fractions obtained after three transfections were analyzed for the indicated proteins by immunoblotting. (B) After each round of transfection total-cell lysates were immunoblotted for IRP1 and β-actin (Upper), and cytosolic fractions were analyzed for enzymatic activities of cytAco relative to LDH (Lower). (C) HeLa cells were depleted of NFS1, NBP35, or CFD1 for a total of 6 d by RNAi. At the second round of transfection at day 3, cells received plasmids encoding either an IRP1-EGFP-TEV-FLAG fusion protein or an EGFP-TEV-FLAG reference protein (EGFP only) and were grown for another 3 d in the presence of ⁵⁵Fe-labeled transferrin. FLAG-tagged proteins were immunopurified, and associated radioactivity was determined relative to either recovered IRP1-EGFP fluorescence (filled bars) or total lysate protein (hatched bars). (D, Upper) IRP1 binding (IRP2 supershift method) to ³²P-labeled IRE (³²P-[CTP] IRE) of human ferritin mRNA in the presence or absence of 1.7% β-mercaptoethanol (β-ME) was quantified by phosphorimaging of a native gel. (Lower) The ratio of IRP1-bound ³²P-IRE probe in the absence and presence of β-ME is an inverse measure for the Fe-S cluster maturation status of IRP1. (E) IRE-binding of IRP2 (IRP1 supershift) was probed as in D (Upper) and quantified (Lower), but in the presence of 0.3% β-ME. (F) After the third round of transfection, total-cell lysates were analyzed for TFR1, H-FT, and β-actin by immunoblotting. (Upper) Representative blots. (Lower) Protein levels were quantitated relative to β-actin. Further, TFR1 expression was functionally assessed by estimating cellular binding of Tf-FITC relative to total cellular protein. All values were normalized to mock-transfected cells (set to 100% and indicated by dashed lines) and are expressed as mean ± SD (n ≥ 3). sh, shRNA; si, siRNA.

Fig. 2.

Depletion of CFD1 impairs the maturation of cytosolic Fe-S proteins. HeLa cells were depleted for CFD1 as in Fig. 1, and DPYD, ABCE1, and GPAT were analyzed for enzyme activity, ⁵⁵Fe incorporation, or protein levels. (A, Upper) DPYD-dependent formation of [4-¹⁴C]-dihydrothymine ([4-¹⁴C]-DHT) from [4-¹⁴C]-thymine ([4-¹⁴C]-T) was determined by TLC and subsequent autoradiography. (Lower) Specific DPYD enzyme activity was calculated from the proportion of [4-¹⁴C]-DHT converted from [4-¹⁴C]-T and is presented relative to LDH activity. (B) DPYD and β-actin levels were examined by immunoblotting (Upper) and quantified (Lower). (C) HeLa cells were depleted of NFS1, NBP35, or CFD1 by RNAi, and incorporation of ⁵⁵Fe into an EGFP-ABCE1-TEV-FLAG fusion protein was determined as in Fig. 1. (D) GPAT levels were examined and quantified as in B. Representative autoradiographs and blots are shown. All values were normalized to mock-transfected cells (set to 100% and indicated by dashed lines) and are expressed as mean ± SD (n ≥ 3). scr, scrambled siRNAs; sh, shRNA; si, siRNA.

Fig. 3.

Depletion of CFD1 impairs assembly on the nuclear Fe-S proteins. HeLa cells were RNAi-depleted for CFD1 or for the indicated ISC or CIA factors as in Fig. 1. (A and B) POLD1 (A) and NTHL1 (B) as well as β-actin as a control were examined by immunoblotting (Upper) and quantified (Lower). The immunoblots show the last round of transfection only. (C) Incorporation of ⁵⁵Fe into a NTHL1-EGFP-TEV-FLAG fusion protein was determined as in Fig. 1. Representative blots are shown. All values were normalized to mock-transfected cells (set to 100% and indicated by the dashed line) and are expressed as mean ± SD (n ≥ 3). scr, scrambled siRNAs; sh, shRNA; si, siRNA.

Fig. 4.

Human CFD1 interacts physically with NBP35 and IOP1 and is required for Fe-S cluster assembly on IOP1. (A) HeLa cells were cotransfected with plasmids encoding human IOP1 fused to a C-terminal HA-tag (HA₃), human NBP35 fused to a C-terminal DsRed2- or myc-tag, or human CFD1 fused to a C-terminal DsRed2- or Strep-tag, as indicated. After 3 d of tissue culture, cleared lysates were subjected to affinity purification by Strep-Tactin, anti-myc, or anti-HA resins. Cleared lysate (50 µg protein, Input) and bead-associated material (one-third of total precipitate) were immunostained for endogenous or tagged IOP1, NBP35, and CFD1. (B) HeLa cells were depleted for CFD1 or NBP35 as in Fig. 1 and were analyzed for CFD1, NBP35, or β-actin levels as indicated. (C) HeLa cells were depleted for CFD1 and analyzed for IOP1 and β-actin levels. (D) HeLa cells were depleted for NFS1, NBP35, or CFD1, and incorporation of ⁵⁵Fe into a IOP1-EGFP-TEV-FLAG fusion protein was determined as in Fig. 1. Representative blots are shown. All values were normalized to mock-transfected cells (set to 100% and indicated by the dashed lines) and are expressed as mean ± SD (n ≥ 3). scr, scrambled siRNAs; sh, shRNA; si, siRNA.

Fig. 5.

Crystal structure of C. thermophilum Cfd1. (A) Cartoon presentation of the crystal structure of dimeric ctCfd1. The left monomer is colored from blue at the N terminus to red at the C terminus. The right monomer is shown in blue and red for α-helices and β-strands, respectively. A sulfate ion near the P-loop is depicted as spheres. A [4Fe-4S] cluster at the dimer interface is indicated by the anomalous scattering contribution of iron at 1.73-Å wavelength (blue meshed map contoured at 8 σ). Conserved cysteine residues are highlighted as sticks with carbon in cyan and sulfur in gold. Stars indicate residues belonging to the symmetry mate. Dashed lines indicate regions with weak electron density. (B) Superposition of ctCfd1 and bacterial nitrogenase Fe-protein NifH (SI Appendix, Table S2). A subunit of ctCfd1 (cyan) with iron ions shown as pink spheres is superposed onto a subunit of the Fe-protein (PDB ID code 4WZB, green) with irons shown as light blue spheres. (C) Schematic comparison of the relative arrangement of Fe-S clusters and ATP in ctCfd1 and NifH.

Fig. 6.

C. thermophilum Cfd1 and Nbp35 form a complex with differential binding preferences for ATP and GTP. Purified recombinant ctCfd1 and ctNbp35 were labeled with the fluorescent dye NT 647 either individually or as complex and were analyzed for ligand binding by thermophoresis. (A) Binding constants (1/K_d) for the interaction between labeled ctNbp35, ctCfd1, or the ctCfd1-ctNbp35 complex and the three nucleotides ATP, GTP, or CTP. (B) Binding constants (1/K_d) for labeled ctNbp35 or ctCfd1 and their nonlabeled protein partners. Values are given as mean ± SD (n ≥ 3). nd, nondetectable.