Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer.
Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-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-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, 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 alpha-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.
Nishino
Tomonori G
TG
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Kitano
Ken
K
Kojima
Katsuhiko
K
Ogishima
Tadashi
T
Ito
Akio
A
Kitada
Sakae
S
D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D000096122:Mitochondrial Processing Peptidase
D005456:Fluorescent Dyes; D005998:Glycine; D049452:Green Fluorescent Proteins; D008239:Lysine; D008666:Metalloendopeptidases; D010447:Peptide Hydrolases; D012694:Serine; D000096122:Mitochondrial Processing Peptidase