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

• Published in: The Journal of biological chemistry 2012-04, Vol.287 (15), p.12365-12378
• Main Authors: Netz, Daili J.A., Pierik, Antonio J., Stümpfig, Martin, Bill, Eckhard, Sharma, Anil K., Pallesen, Leif J., Walden, William E., Lill, Roland
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - chemistry; Amino Acid Motifs; Amino Acid Substitution; Binding Sites; Biosynthesis; Cofactor; Conserved Sequence; Coordination Complexes; Cysteine - genetics; Electron Paramagnetic Resonance (EPR); Enzyme Mutation; Enzymology; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Guanosine Triphosphate - chemistry; Iron; Iron-Sulfur Protein; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Mossbauer Spectroscopy; Mutagenesis, Site-Directed; Oxidation-Reduction; P-loop NTPase; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sulfur; Yeast Genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.328914

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. Background: The P-loop NTPases Cfd1 and Nbp35 form a scaffold complex for cytosolic-nuclear Fe-S cluster synthesis. Results: Two C-terminal cysteine residues of each Cfd1 and Nbp35 assemble a transient [4Fe-4S] cluster in a nucleotide-dependent fashion. Conclusion: The results suggest a bridging nature of the transient [4Fe-4S] cluster of Cfd1-Nbp35. Significance: This study defines the molecular role of the Cfd1-Nbp35 tetrameric scaffold complex in Fe-S cluster assembly.