ABCC1 p.Lys31Ala
| Predicted by SNAP2: | A: N (82%), C: N (53%), D: N (78%), E: N (87%), F: N (61%), G: N (72%), H: N (87%), I: N (82%), L: N (82%), M: N (72%), N: N (87%), P: N (78%), Q: N (93%), R: N (93%), S: N (93%), T: N (87%), V: N (82%), W: D (63%), Y: D (66%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] A bridging [4Fe-4S] cluster and nucleotide binding... J Biol Chem. 2012 Apr 6;287(15):12365-78. Epub 2012 Feb 23. Netz DJ, Pierik AJ, Stumpfig M, Bill E, Sharma AK, Pallesen LJ, Walden WE, Lill R
A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation.
J Biol Chem. 2012 Apr 6;287(15):12365-78. Epub 2012 Feb 23., [PMID:22362766]
Abstract [show]
The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.
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No. Sentence Comment
259 First, yeast cells bearing only the Walker A-mutated Cfd1 (K31A) or Nbp35 (K86A), generated by plasmid FIGURE 6.
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ABCC1 p.Lys31Ala 22362766:259:59
status: NEW312 C, 55 Fe incorporation into plasmid-encoded Cfd1-TAP or its K31A mutant version was measured in Gal-CFD1 cells grown in galactose- or glucose-containing minimal medium as indicated.
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ABCC1 p.Lys31Ala 22362766:312:60
status: NEW311 C, 55 Fe incorporation into plasmid-encoded Cfd1-TAP or its K31A mutant version was measured in Gal-CFD1 cells grown in galactose- or glucose-containing minimal medium as indicated.
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ABCC1 p.Lys31Ala 22362766:311:60
status: NEW[hide] The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster... J Biol Chem. 2015 Sep 25;290(39):23793-802. doi: 10.1074/jbc.M115.667022. Epub 2015 Jul 20. Camire EJ, Grossman JD, Thole GJ, Fleischman NM, Perlstein DL
The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.
J Biol Chem. 2015 Sep 25;290(39):23793-802. doi: 10.1074/jbc.M115.667022. Epub 2015 Jul 20., [PMID:26195633]
Abstract [show]
Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 muM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.
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No. Sentence Comment
138 Lane 1, His Nbp35; lane 2, His Cfd1; lane 3, Nbp35-Cfd1 complex; lane 4, Nbp35-K26A Cfd1; lane 5, Nbp35-K31A Cfd1; lane 6, K86A Nbp35-Cfd1.
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ABCC1 p.Lys31Ala 26195633:138:104
status: NEW150 The lysine mutants K86A Nbp35-Cfd1, Nbp35-K26A Cfd1, and Nbp35-K31A Cfd1 were expressed at similar levels as the wild-type Nbp35-Cfd1 complex, whereas little K81A Nbp35 was present in crude extracts upon co-expression with wild-type Cfd1.
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ABCC1 p.Lys31Ala 26195633:150:63
status: NEW151 For the remaining three mutants, intact complexes containing K86A Nbp35-Cfd1, Nbp35-K26A Cfd1, and Nbp35-K31A Cfd1 could be obtained.
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ABCC1 p.Lys31Ala 26195633:151:105
status: NEW154 To confirm via a second independent method that our mutations did not significantly affect the structure of the heterocomplex, we analyzed the secondary structure content of the two canonical lysine mutants, K86A Nbp35-Cfd1 and Nbp35-K31A Cfd1, via CD.
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ABCC1 p.Lys31Ala 26195633:154:234
status: NEW202 C shows wild-type Nbp35-Cfd1 complex (afb;), Nbp35-K26A Cfd1 (); Nbp35-K31A Cfd1(Éa;), and Nbp35-D55A Cfd1 (E).
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ABCC1 p.Lys31Ala 26195633:202:80
status: NEW220 We found that mutation of either lysine in the Walker A motif, K26A or K31A, resulted in decreased ATP hydrolysis activity of the Nbp35-Cfd1 complex (Fig. 5C, triangles).
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ABCC1 p.Lys31Ala 26195633:220:71
status: NEW