title: Glycine-rich loop of mitochondrial processing peptidase alpha-subunit is responsible for substrate recognition by a mechanism analogous to mitochondrial receptor Tom20.
abstract: 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.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Klára
authors.initials: K
authors.lastname: Dvoráková-Holá
authors.forename: Anna
authors.initials: A
authors.lastname: Matusková
authors.forename: Martin
authors.initials: M
authors.lastname: Kubala
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
authors.forename: Tomás
authors.initials: T
authors.lastname: Kucera
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Vecer
authors.forename: Petr
authors.initials: P
authors.lastname: Herman
authors.forename: Natalya
authors.initials: N
authors.lastname: Parkhomenko
authors.forename: Eva
authors.initials: E
authors.lastname: Kutejova
authors.forename: Jirí
authors.initials: J
authors.lastname: Janata
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005454:Fluorescence Polarization; D005998:Glycine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D000096122:Mitochondrial Processing Peptidase
chemical_list: D004271:DNA, Fungal; D005998:Glycine; D008666:Metalloendopeptidases; D033681:Mitochondrial Membrane Transport Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000096122:Mitochondrial Processing Peptidase; C489350:TOM20 protein, S cerevisiae