Fig 1: Fe–S cluster incorporation into nsp13 occurs through its interactions with components of the Fe–S biogenesis machinery. (A) Domain organization of nsp13. The ZBD is shown in dark blue, followed by a “stalk” region (light blue), a ß-barrel, 1B, in magenta, and two helicase core domains RecA1 (1A) and RecA2 (A2) (19). (B) Structure of the N-terminal zinc-binding domain (ZBD) of nsp13 showing the amino acid residues ligating zinc in the available crystal structure (PDB ID: 6ZSL) (20). A completely conserved LYR-like motif (LYK) in the seven human pathogenic coronaviruses is highlighted in yellow. (C) Proposed model of the chaperone/cochaperone-mediated transfer of nascent Fe–S clusters, assembled on the main scaffold protein, ISCU, through the direct binding of the cochaperone HSC20 to LYR motifs present in recipient apoproteins. ATP hydrolysis by the HSC20-cognate chaperone, HSPA9, is proposed to facilitate cluster transfer to recipient proteins, while concomitantly driving folding of the recipient protein into its final conformation. (D) Multiple sequence alignment of the residues ligating zinc and of the conserved LYR-like motif in nsp13 of the seven human pathogenic coronaviruses. Boxes in the same color identify the amino acid residues in each individual metal-binding site. (E) Representative Coomassie blue staining of pull-down assays performed with purified proteins. Purified nsp13-Strep II (0.25 µg) or the variants wherein either the LYK motif or the cysteine and histidine residues coordinating zinc in the available structures were replaced by alanines or serines, respectively, as indicated, were combined with 0.25 µg of HSC20. Immunoprecipitations (IPs) were performed with Strep II antibody to immunocapture nsp13 proteins. The presence of HSC20 (a.k.a. HSCB) in the eluates after IPs of nsp13 proteins was analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie staining. Aliquots corresponding to 20% of the inputs were run on the gel for comparison (n = 4 biological replicates). (F) Eluates after IPs of nsp13 wild type (WT) or variants recombinantly expressed in 293T cells, as indicated, were probed with antibodies against Strep II to verify the efficiency of IP and against components of the Fe–S cluster (HSC20, HSPA9, and NFS1) and of the cytoplasmic Fe–S (CIA) assembly machinery (CIAO1, MMS19, and FAM96B). Immunoblots to PCBP1 and BOLA2 are also shown as these proteins were identified in our mass spectrometry analysis and have been reported to deliver iron for cytoplasmic Fe–S cluster assembly (21) (n = 4).
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