The HsPDF asymmetric unit is constituted by two HsPDF dimers. (a) Stereo ribbon view of the HsPDF dimer. Each monomer is shown in a different color, with the N-terminus and C-terminus indicated as N-term, and C-term, respectively. The Co²⁺ atom is a purple sphere. (b) The dimer interface is formed through electrostatic and hydrophobic interactions between the HsPDF monomers. Stereo view, the carbon backbone of each of the monomers is in a different color, green or cyan, with nitrogen atoms in blue, oxygen atoms in red, and sulfur atoms in yellow. Salt bridges are indicated by gray dotted lines. (c) A 2D depiction of the dimer interface in (b). Residues in blue and black correspond to each of the interacting HsPDF monomers. Residues in gray correspond to the monomer in black, and are located in a different plane, at the core of the dimer interface.

The active site metal, Co²⁺, is coordinated to four ligands, three of them residues of HsPDF and a fourth unexpected ligand. (a) The geometry of the metal in HsPDF is close to tetrahedral. Bond angles between the active site Co²⁺ and the residues in HsPDF that coordinate it are shown. A representation similar to that reported for EcPDF55 is shown for ease of comparison. (b) The fourth metal coordinating ligand in the HsPDF structure model is a phosphate molecule. Electron density of the HsPDF active site is shown in stereo view. F_o–F_c is contoured at 5σ in yellow. The four ligands of the Co²⁺ atom, in purple, are H156, H160, C114, and a fourth molecule modeled as phosphate, in orange. The distances of the presumed oxygen atoms in the phosphate molecule to neighboring atoms are shown by dotted lines. (c) Diagram depiction of the metal coordination site in HsPDF, including surrounding residues at the active site. Carbon, oxygen, nitrogen, sulfur and phosphorus atoms are shown as black, red, blue, yellow and green spheres, respectively. Distances (in Å) between atoms are depicted as broken green lines and labels. Atom labels are in black. The diagram was generated with LigPlot v.4.4.2.56

Identification of the HsPDF substrate-binding site in the actinonin-bound HsPDF structure model. (a) Structure of the peptidomimetic inhibitor actinonin with assigned atom numbers. (b) Stereo image of the active site of HsPDF with actinonin. HsPDF residues, in gray, contacting actinonin, yellow, are shown as sticks. Hydrogen bonding is represented by black dotted lines with interatomic distances also shown. (c) Ball and stick 2D diagram of actinonin interactions at the HsPDF active site. Hydrogen bonds at six points of actinonin with corresponding donors/acceptors in HsPDF are denoted as areas A–E, in light blue. Distances (in Å) between the hydrogen bonding atoms are shown in green and denoted by broken green lines. Regions of hydrophobic interaction between actinonin and HsPDF are shown as light blue regions F–H. Hydrophobic interactions of actinonin with HsPDF are shown as red radiating lines surrounding the atoms, with their respective partner in HsPDF also indicated by name and red radiating lines. Interactions at the A–H regions in non-mammalian PDFs are described in detail in Supplementary Data Table 1.

There is no S2′ binding pocket in HsPDF. Actinonin-bound HsPDF and virtual peptide-binding modeling, all shown in the same orientation, indicate lack of an S2′ binding pocket. Despite the lack of an S3′ substrate-binding pocket, actinonin-bound HsPDF and the virtual models suggest that residues in HsPDF surrounding P3′ of a substrate might influence substrate specificity. (a) Actinonin-bound HsPDF stereo image. Actinonin, yellow sticks, interacts with HsPDF, in gray. Residues in HsPDF surrounding P2′ of actinonin are shown as sticks. Distances (in Å) between the valine side chain in actinonin and the closest residues in HsPDF, G113 and R48, are shown as dotted pink lines. (b) Actinonin-bound HsPDF surface view in stereo mode. The image represents the same structure and orientation as in a, but in surface view. (c) Met-Thr-His-Gln, MTHQ, bound HsPDF virtual model. MTHQ, in cyan, was modeled into HsPDF based on the actinonin-bound HsPDF structural model using the program CNS.50 (d) Met-Phe-Ala-Asp, MFAD, bound HsPDF virtual model. MFAD, in green, was modeled into HsPDF based on the actinonin-bound HsPDF structural model. Modeling was done as in c.

N-Met-formylated peptides derived from the N-terminus of mitochondrial DNA-encoded proteins are substrates of HsPDF in vitro. Apparent K_m and k_cat were measured by a formate dehydrogenase, FDH, NAD⁺-coupled assay (see Materials and Methods). A representative curve for the processing of three substrates is shown. Values represent the mean rate of reaction (in μmol NADH min⁻¹ mg⁻¹ of HsPDF) ± S.E.M. for triplicate samples for each concentration of substrate. (a) HsPDF kinetics with formyl-Met-Ala-His-Ala, fMAHA. (b) HsPDF kinetics with formyl-Met-Asn-Phe-Ala, fMNFA. (c) HsPDF kinetics with formyl-Met-Met-Tyr-Ala, fMMYA.

HsPDF has the conserved PDF fold observed in other non-mammalian PDFs. (a) Protein sequence alignment of HsPDF to non-mammalian PDFs: Escherichia coli PDF, (EcPDF, Genbank protein ID gi15803814), Staphylococcus aureus PDF (SaPDF, Genbank protein ID gi22219286), and Arabidopsis thaliana PDF (AtPDF, Genbank protein ID gi11320952). HsPDF and AtPDF sequences both belong to N-terminal truncated PDFs. Alignment is colored according to the percentage identity of residues among the sequences. Dark blue, >80%; blue, >60%; light blue, >40%; and no color, <40%. Alignment was generated with ClustalW53 and Jalview.54 (b) Protein sequence alignment of HsPDF to predicted mammalian PDFs. PDF of Bos taurus (BtPDF Genbank ID XP_001255524.1), Canis familiaris (CfPDF Genbank ID XP_853641.1), Rattus norvegicus (RnPDF Genbank ID XP_001073696.1), and Mus musculus (MmPDF Genbank ID AK075957.1). Sequence alignment was done as described for a. (c) Stereo view of HsPDF monomer. Secondary structure elements are numbered. H, α Helix; S, β strand. The Co²⁺ atom is a purple sphere. Regions of HsPDF with structural similarity to non-mammalian PDFs are shown in gray.

Differences between HsPDF and non-mammalian PDFs. HsPDF shares the β hairpin loop anchoring points that shape the entrance to the active site in the PDF family. However the conformation of the C-terminus in HsPDF, in combination with the presence of the H2/H3 α-helical loop creates a characteristic entrance to its active site compared to non-mammalian PDFs. The HsPDF Co²⁺ metal is represented as a purple/pink sphere in all panels. Structural alignments in b–d were done with PyMol. Residues included in the C^α RMSD calculations in these panels are shown as gray regions in HsPDF in Fig. 2. RMSD values were obtained with the program O.51 Structures in b–h are shown in the same orientation. (a) Stereo representation of the HsPDF dimer, highlighting the S5/S6 hairpin anchoring points. Hairpin anchoring at its N-terminal base by residues on H3 and H4 are shown in cyan, anchoring at the crest of the hairpin by residues on H1, the N-terminus, and at the dimer interface are shown in green, and anchoring at the C-terminal side of the hairpin by residues at the C-terminus of HsPDF are shown in magenta. (b) HsPDF, red in b–d, aligned to type 1B EcPDF (PDB entry 1BS7) green.55 RMSD is 1.252 Å for the C^α atom alignment of the HsPDF/EcPDF pair. (c) HsPDF alignment to type 2 SaPDF (PDB entry 1LQW),23 blue. RMSD is 1.314 Å for the C^α atom alignment of the HsPDF/SaPDF pair. (d) HsPDF alignment to type 1A AtPDF monomer (PDB entry 1ZXZ),31 cyan. The RMSD is 0.917 Å for the C^α atom alignment of the HsPDF/AtPDF pair. (e) HsPDF active site entrance. (e–h) Surface views of PDF from various organisms in gray, with oxygen atoms in red, nitrogen atoms in blue and sulfur atoms in yellow. The PDF structures correspond to the same PDB entries as those in b and c. (f) EcPDF, a PDF type 1B, active site entrance. (g) SaPDF, a type II PDF, active site entrance. (h) AtPDF, a representative plant type 1A PDF, active site entrance.

The substrate-binding pockets of HsPDF differ from those of EcPDF and AtPDF. (a) The S1′ binding pocket in HsPDF is narrower than that of EcPDF. The upper stereo pair shows the HsPDF S1′ pocket with the protein residues in gray, and the actinonin molecule in yellow. The bottom stereo pair shows the same pocket for EcPDF (1G2A),32 also in gray. (b) HsPDF has no well-defined S3′ pocket. AtPDF has an S3′ pocket formed by the residues shown in the bottom stereo pair, where the side chain of the product MAS, yellow, fits, (PDB 1ZY1). However, the equivalent depression in HsPDF is less concave, upper stereo pair. The MAS product was modeled from that of AtPDF (PDB 1ZY1).