| Human peptide deformylase, HsPDF, removes the N-terminal formyl-modification on the methionine of peptide substrates in an in vitro assay, analogous to the activity of its E. coli counterpart, EcPDF. EcPDF deformylates nascent proteins emerging from the ribosome. The cellular function of HsPDF is controversial, mainly because of its poor catalytic efficiency compared to the non-mammalian counterparts. Therefore whether HsPDF fulfills a cellular function is unknown. Previous studies have shown that HsPDF localizes to the mitochondria and that pharmacologic and genetic downregulation of its activity in cancer cells result in loss of cell proliferation and viability. To enable discovery of novel HsPDF targeted drugs, we determined the crystal structure of HsPDF alone and in complex with the inhibitor actinonin, and elucidated the cellular function of HsPDF.;Here we show that HsPDF can deformylate its putative substrates, derived from mitochondrial DNA encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however shape a characteristic active site entrance. The actinonin bond HsPDF (1.7 A) identified the substrate binding site. A defined S1', but no S2' and S3' substrate binding pockets exists. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggests a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover.;This work also shows that HsPDF is necessary for the accumulation of functional mtDNA-encoded proteins. Inhibition of HsPDF reduces mitochondrial-encoded proteins, respiratory function, and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. HsPDF inhibition also causes transcriptional upregulation of integrated/ER stress response markers. HsPDF inhibition, but not inhibition of mitochondrial translation by chloramphenicol, severely decreases mitochondrial membrane potential (Psimt), which may contribute to metabolic catastrophe leading to loss of proliferation and viability in cancer cells. We show that unprocessed mitochondrial proteins, rather than lack of these proteins, cause cytotoxicity, and that deformylation of mtDNA-encoded proteins occurs in the mitochondria. |
