Substrate evokes translocation of both domains in the mitochondrial processing peptidase alpha-subunit during which the C-terminus acts as a stabilizing element.
All three tryptophan residues in alpha-subunit of mitochondrial processing peptidase (MPP) were subsequently substituted. While substitutions of Trp223 led to misfolded non-functional protein, mutations of Trp147 and/or Trp481 did not affect the enzyme processing activity. Thus, fluorescence properties of the mutants with fewer tryptophans were used for observation of both alpha-MPP domain translocation and visualization of conformational changes in the interdomain linker evoked by substrate. We found that in the presence of substrate the C-terminal penultimate Trp481 was approaching Trp223, which is localized at the border of N-terminal domain and interdomain linker. Also, excision of the alpha-MPP C-terminal 30 amino acid residues (DeltaC30) led to a complete loss of protein function. Even shorter deletions of the alpha-MPP C-terminus destabilized the protein slightly (DeltaC2) or dramatically (DeltaC17). It suggests that the extreme C-terminus of alpha-MPP provides mechanical support to the C-terminal domain during its extensive conformational change accompanying the substrate recognition process.
Janata
Jirí
J
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. janata@biomed.cas.cz
Holá
Klára
K
Kubala
Martin
M
Gakh
Oleksandr
O
Parkhomenko
Natalya
N
Matusková
Anna
A
Kutejová
Eva
E
Amler
Evzen
E
D019943:Amino Acid Substitution; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D017384:Sequence Deletion; D014364:Tryptophan / Q000235:genetics; D000096122:Mitochondrial Processing Peptidase
D005656:Fungal Proteins; D008666:Metalloendopeptidases; D011498:Protein Precursors; D021122:Protein Subunits; D014364:Tryptophan; D000096122:Mitochondrial Processing Peptidase