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

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) α-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast α-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 α-MPP with a single-residue deletion in the loop (Y303W/ΔG292) 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 α-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 α-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.