Spatial Orientation of Mitochondrial Processing Peptidase and a Preprotein Revealed by Fluorescence Resonance Energy Transfer

• Published in: Journal of biochemistry (Tokyo) Vol. 141; no. 6; pp. 889 - 895
• Main Authors: Nishino, Tomonori G, Kitano, Ken, Kojima, Katsuhiko, Ogishima, Tadashi, Ito, Akio, Kitada, Sakae
• Format: Journal Article
• Language: English
• Published: England Japanese Biochemical Society 01.06.2007; Oxford University Press
• Subjects: Amino Acid Sequence; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - pharmacology; Glycine - chemistry; Green Fluorescent Proteins - metabolism; Lysine - chemistry; Metalloendopeptidases - chemistry; Mitochondria - metabolism; Mitochondrial Processing Peptidase; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Peptide Hydrolases - metabolism; Saccharomyces cerevisiae - metabolism; Serine - chemistry; GFP; protease; processing; FRET; mitochondria
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvm095

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric α-MPP and β-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser⁸⁴ and -Lys¹⁵⁶ of β-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 Å, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of α-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.