A proposed common structure of substrates bound to mitochondrial processing peptidase.
Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha- and beta-subunits, specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Structural information of the substrate bound to MPP was obtained using fluorescence resonance energy transfer (FRET) measurement. A series of the peptide substrates, which have distal arginine residues required for effective cleavage at positions -7, -10, -14, and -17 from the cleavage site, were synthesized and covalently labeled with 7-diethyl aminocoumarin-3-carboxylic acid at the N termini and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) at position +4, as fluorescent donor and acceptor, respectively. When the peptides were bound to MPP, substantially the same distances were obtained between the two probes, irrespective of the length of the intervening sequence between the two probes. When 7-diethylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin was introduced into a single cysteine residue in beta-MPP as a donor and IANBD was coupled either at the N terminus or the +4 position of the peptide substrate as an acceptor, intermolecular FRET measurements also demonstrated that distances of the donor-acceptor pair were essentially the same among the peptides with different lengths of intervening sequences. The results indicate that the N-terminal portion and the portion around the cleavage site of the presequence interact with specific sites in the MPP molecule, irrespective of the length of the intervening sequence between the two portions, suggesting the structure of the intervening sequence is flexible when bound to the MPP.
Kojima
K
K
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Kitada
S
S
Ogishima
T
T
Ito
A
A
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D004735:Energy Transfer; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D000096122:Mitochondrial Processing Peptidase
D001120:Arginine; D008666:Metalloendopeptidases; D000096122:Mitochondrial Processing Peptidase