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.

Download full-text


Available from: Tomáš Kučera,
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 09/2013; 8(9):e74518. DOI:10.1371/journal.pone.0074518 · 3.23 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial targeting sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. Since only α-MPP was duplicated in Neurospora, its single β-MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins showed that trypanosomal N-MTSs were comparable to N-MTSs from other organisms. N-MTS cleavage is mediated by a standard heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential for the parasite. cp1 and cp2 are encoded by distinct genes, and in the bloodstream forms the expression of cp1 is downregulated along with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, while cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not appear to have an ancient character proposed earlier for this eukaryote.
    Genome Biology and Evolution 04/2013; 5(5). DOI:10.1093/gbe/evt056 · 4.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Conventional molecular dynamics simulations on 50 ns to 1 μs time scales were used to study the effects of explicit solvent models on the conformational behavior and solvation of two oligopeptide solutes: α-helical EK-peptide (14 amino acids) and a β-hairpin chignolin (10 amino acids). The widely used AMBER force fields (ff99, ff99SB, and ff03) were combined with four of the most commonly used explicit solvent models (TIP3P, TIP4P, TIP5P, and SPC/E). Significant differences in the specific solvation of chignolin among the studied water models were identified. Chignolin was highly solvated in TIP5P, whereas reduced specific solvation was found in the TIP4P, SPC/E, and TIP3P models for kinetic, thermodynamic, and both kinetic and thermodynamic reasons, respectively. The differences in specific solvation did not influence the dynamics of structured parts of the folded peptide. However, substantial differences between TIP5P and the other models were observed in the dynamics of unfolded chignolin, stability of salt bridges, and specific solvation of the backbone carbonyls of EK-peptide. Thus, we conclude that the choice of water model may affect the dynamics of flexible parts of proteins that are solvent-exposed. On the other hand, all water models should perform similarly for well-structured folded protein regions. The merits of the TIP3P model include its high and overestimated mobility, which accelerates simulation processes and thus effectively increases sampling.
    Journal of Chemical Theory and Computation 11/2010; 6(11):3569-3579. DOI:10.1021/ct1003687 · 5.50 Impact Factor
Show more