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.
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. janata@biomed.cas.cz
Jirí
J
Janata
Klára
K
Holá
Martin
M
Kubala
Oleksandr
O
Gakh
Natalya
N
Parkhomenko
Anna
A
Matusková
Eva
E
Kutejová
Evzen
E
Amler
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