Glycine-Rich Loop of Mitochondrial Processing Peptidase α-Subunit Is Responsible for Substrate Recognition by a Mechanism Analogous to Mitochondrial Receptor Tom20

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
Journal of Molecular Biology (Impact Factor: 4.33). 03/2010; 396(5):1197-210. DOI: 10.1016/j.jmb.2009.12.054
Source: PubMed


Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) alpha-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast alpha-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility. Moreover, time-resolved measurements showed that a functionless alpha-MPP with a single-residue deletion in the loop (Y303W/DeltaG292) is defective particularly in the primary contact with substrate. Thus, the GRL was proved to be part of a contact site of the enzyme specifically recognizing the substrate. Regarding the surface exposure and presence of the hydrophobic patches within the GRL, we proposed a functional analogy between the presequence recognition by the hydrophobic binding groove of the Tom20 mitochondrial import receptor and the GRL of the alpha-MPP. A molecular dynamics (MD) simulation of the MPP-substrate peptide complex model was employed to test this hypothesis. The initial positioning and conformation of the substrate peptide in the model fitting were chosen based on the analogy of its interaction with the Tom20 binding groove. MD simulation confirmed the stability of the proposed interaction and showed also a decrease in GRL flexibility in the presence of substrate, in agreement with fluorescence measurements. Moreover, conserved substrate hydrophobic residues in positions +1 and -4 to the cleavage site remain in close contact with the side chains of the GRL during the entire production part of MD simulation as stabilizing points of the hydrophobic interaction. We conclude that the GRL of the MPP alpha-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.

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Available from: Tomáš Kučera
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    • "The variability in phenotype could potentially be explained by a different functional impact of the mutations. The previously reported p.Ala377Thr mutation is in close proximity to the glycine-rich loop, which is the most conserved part of MPPa and is crucial for initial interaction with and binding to the substrate (Nagao et al., 2000; Dvorakova-Hola et al., 2010; Kucera et al., 2013). Jobling et al. (2015) showed that the mutation resulted in a decreased amount of MPPa in patients and that it altered the processing of frataxin. "

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    • "Although the MPP crystal structure was published more than a decade ago [4], very few studies have addressed the detailed operation of MPP. To date, the substrate binding and cleavage mechanism in the MPP active site (AS) has been described in detail [19] (AS-bound structure in Figure 1) and a mechanism of substrate recognition by the GRL has been outlined [17] (GRL-bound structure in Figure 1), however the mechanism of substrate translocation from the GRL to the MPP active site has not yet been investigated. Since the whole process is complex and involves interactions of larger regions rather than single amino acids, the experimental approach using amino acid point mutations is problematic. "
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    ABSTRACT: An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.
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    • "MPP is a zinc-dependent metallopeptidase that consists of two homologous subunits, a and b, which are both required for its processing activity (Schneider et al. 1990; Geli 1993). The a-MPP subunit possesses a highly conserved glycine-rich loop required for the recognition of N-MTSs (Nagao et al. 2000; Dvorakova-Hola et al. 2010), whereas the metal-binding motif HXXEHX n E of b-MPP is the catalytic site responsible for cleavage of the peptide bond (Kitada et al. 1995; Striebel et al. 1996). In various eukaryotes, such as metazoans and Saccharomyces cerevisiae, MPP is present in the mitochondrial matrix as a general presequence processing enzyme. "
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