Figure 3. V. radiata’s CIII₂ mitochondrial processing peptidase (MPP) domain has a conserved architecture and active site but contains plant-specific secondary-structure elements not seen in other CIII-MPP subunits or in soluble MPP.

(A) Ribbon representation of the VrMPP-α (blue) and VrMPP-β (light blue) looking into the central cavity. Dashed rectangle indicates the location of the active site, detailed in (B). (B) MPP-β active site [rotated 90° about vertical axis with respect to (A)]. Shown in stick representation are the Zn-coordinating residues (His137, His141, Glu217), the catalytic water-activating residue (Glu140) and conserved, putative substrate-recognition residues (Phe144, Glu227, Asp231, Asn167, Tyr169). Residue Ala168 is also conserved, but not visible in this orientation. (C–D) Superposition of V. radiata and S. cerevisiae CIII₂ MPP domain subunits. VrMPP-α and -β’s structural elements not present in ScCor2 and ScCor1 are marked. Structural elements that are additionally not present in yeast soluble MPP, i.e. plant-specific features, are marked with a cross (†). (D) VrMPP-α (blue) and ScCor2 (dark pink). (D) VrMPP-β (light blue) and ScCor1 (dark pink). β, β−strand; α, α-helix; N-ter, N-terminus. See also Figure 3—figure supplements 1–2 for further details. S. cerevisiae structures from PDB: 6HU9.

Figure 3—figure supplement 1. Further characterization of VrMPP subunits and their homologs.

(A-B) Electrostatic potential of surface of VrMPP-β (A) and VrMPP-α (B). Electrostatic potential was calculated using Delphi (Li et al., 2012; Li et al., 2019), with standard parameters. Red, negative; white, neutral; blue, positive. (C) VrMPP-β-anchoring interactions. The main anchor is marked with an asterisk (*) and comprises the β-sheet with strands from VrMPP-β, VrCYC1 and VrQCRC, as well as further interactions with the VrUCR1 N-terminus and helix from VrQCR7. The plant-specific interactions joining one MPPα/β dimer to the other (N-termini of VrMPP-α and VrMPP-β) are marked with a cross (†). (D) Sequence identity percentages between MPP-β and MPP-α homologs both CIII₂ subunits and soluble MPP in V. radiata (Vr), S. cerevisiae (Sc) and B. taurus (Bt). MPP subunits that are part of CIII and that possess expected MPP enzymatic activity are marked with a plus symbol (MPP activity could not be confirmed for our isolated sample—see main text). A plus symbol in parenthesis indicates that BtUQCR1/2 only have known enzymatic MPP activity towards one substrate. Sequence identity percentages were calculated using the Clustal Omega multiple alignment tool in Geneious. (E) Protein sequence alignment of VrMPP-β subunit (VrMPPb) and its CIII₂ and soluble MPP homologs from (D) were aligned with Clustal Omega. For space, only the first ~200 residues of each sequence are shown. Key residues and sequences are highlighted as follows. Green: catalytic residues; orange: substrate-recognition residues; gray: cleaved import sequence (as per Uniprot entry and/or existing structure); yellow: VrMPP-β’s N terminal extension. Symbols underneath aligned residues: * fully conserved, : conservation between group of strongly similar properties, . conservation between group of weakly similar properties.

Figure 3—figure supplement 2. Alignment of MPP-α homologs.

(A) Protein sequences of V. radiata‘s MPP-α subunit (VrMPPa), S. cerevisiae (Sc) and B. taurus (Bt) CIII₂ (Cor2, UQCR2) and soluble MPP (MPPa) subunits were aligned with Clustal Omega. Key residues and sequences are highlighted as follows: gray for cleaved import pre-sequence (as per Uniprot entry and/or existing structure); yellow for VrMPP-α’s N terminal extension; green for substrate binding/release glycine-rich loop; blue for elements missing in ScCor2; orange for VrMPP-α’s plant-specific additional secondary-structure elements. Symbols underneath aligned residues: * fully conserved, : conservation between group of strongly similar properties, . conservation between group of weakly similar properties. (B) Summary of differences in structural features of VrMPP-α and its homologs from (A), with the same color coding. Plus symbols (+) indicate the presence of the structural element (residue numbers in parenthesis), obtained from inspection of the structures. MPP subunits that are part of CIII and that possess expected MPP enzymatic activity are marked with a plus symbol (MPP activity could not be confirmed for our isolated sample—see main text). PDB codes of structures used: 1HR6 (ScMPP-α), 6HU9 (ScCor2), 1BGY (BtUQCR2). N.a. indicates that the BtMPP-α structure is not available and thus the comparison could not be made.