Crystal structure of Escherichia coli YfhJ protein, a member of the ISC machinery involved in assembly of iron–sulfur clusters

Introduction.Iron-sulfur (Fe-S) proteins are ubiquitously distributed in organisms, playing essential roles in various biological processes as electron carriers, enzymes, and sensors of oxygen and iron.[1][2][3] These functions depend on the chemical versatility of their cofactors, Fe-S clusters, which consist of several iron and sulfur atoms.Recent genetic and biochemical analyses have revealed that several enzymatic systems are involved in vivo in the synthesis of Fe-S clusters and in their incorporation into apo Fe-S proteins; three Fe-S cluster assembly machineries (NIF, ISC, and SUF) have so far been identified.In the NIF system, NifS and NifU are required for the formation of metalloclusters of nitrogenase in Azotobacter vinelandii. 4 -6 Furthermore, the NIF-like machinery has been shown to work in the maturation of a wide variety of Fe-S proteins.7 The ISC machinery, discovered from the homology between NifS and IscS, 6,8 has broad specificity and targets general Fe-S proteins.9,10 The third SUF machinery was identified as an alternative pathway of Fe-S cluster assembly in Escherichia coli.11 The ISC machinery is present in ␣-, ␤-, and ␥-proteobacteria and the mitchondria of eukaryotes, whereas the SUF machinery is widely distributed among archaebacteria, eubacteria, and the plastids of eukaryotes.7 The components of the ISC machinery are encoded in the so-called isc operon (iscRSUA-hscBA-fdx-yfhJ).This machinery was unambiguously demonstrated to be involved in the Fe-S cluster assembly; the overexpression of the isc operon increases the production of recombinant Fe-S proteins, whereas mutation of the operon decreases the activity of Fe-S proteins.9,10,12 IscS is a cysteine desulfurase, which releases a sulfur atom from L-cystein and serves as a sulfur donor for the Fe-S cluster assembly reaction.6,8,13 IscU and IscA bind unstable Fe-S clusters during the in vitro reconstitution process and appear to function as scaffolds in the Fe-S cluster assembly process.14,15 HscA is a molecular chaperone 16 and HscB is a co-chaperone.17 Fdx is a [2Fe-2S]-type ferredoxin.18 IscR is a transcription factor that regulates expression of the isc genes.19 YfhJ (also called ORF3 or IscX) is a small, acidic protein of 66 residues, and pI ϭ 3.7.The homologs of YfhJ are distributed in proteobacteria categorized in the ␤and ␥-subdivisions, in which the gene order (iscRSUA-hscBAfdx-yfhJ) is highly conserved in the respective chromosomes as well as the acidic character of the protein.Two-hybrid and pull-down experiments have shown a specific interaction between YfhJ protein and IscS, 20 indicating that YfhJ protein is involved in the ISC function.However, there is limited information concerning how YfhJ protein functions, since the mutation of the yfhJ gene does not show any conspicuous phenotype.12 Here we report a crystal structure of YfhJ from E. coli at 1.75 Å resolution and the characteristic charge distribution on its protein surface. Materials and Methods.Overproduction and purification: The coding region of YfhJ was amplified by polymerase chain reaction (PCR) with the primers 5Ј-GGATCCGGACT-TAAGTGGACCGATAG-3Ј and 5Ј-GTCGACTATTATTCGG-CCTCGTCCAGC-3Ј, where the underlined sequences are restriction sites of BamHI and SalI, respectively.The PCR product was cloned into the pCR2.1-TOPOvector (Invitrogen) using a TA cloning method, and the sequence was verified.The plasmid was digested with BamHI and SalI, and the fragment was cloned into the corresponding sites of the pQE-30 vector (Qiagen) to construct the pQ-ORF3 plasmid expressing the N-terminal (His) 6 -tagged YfhJ protein.Synchrotron experiments were performed with the approval of JASRI (2001A0521-NL-np).