The Molecular Recognition Mechanism for Superoxide Dismutase Presequence Binding to the Mitochondrial Protein Import Receptor Tom20 from Oryza sativa Involves an LRTLA Motif

Abstract

Most mitochondrial proteins are synthesized in the cytosol as precursor and imported into the mitochondria by Tom complexes (translocase of outer membrane complexes). Knowledge of the binding mechanism between precursor and Tom20 in plants is very limited. Here, computational methods are employed to improve our understanding of the interactions between both molecules. To this end, we model mitochondrial superoxide dismutase precursor (pSOD) in complex with Tom20 in Oryza sativa (OsTom20). In a first stage, five main binding modes were generated using clustering analysis, energy minimization, and expert knowledge. In a second stage, the quality and validity of the resulting complexes is assessed by molecular dynamics (MD) simulations with a generalized Born solvation model. The change in binding free energies is estimated using a computational alanine scanning technique. We identified a particularly favorable complex between pSOD and OsTom20, exhibiting the lowest binding free energy among all candidates and correlating well with experimental data. Furthermore, three independent explicit solvent MD simulations of this structure, each of 100 ns duration, reveal that hydrophobic interactions occur between pSOD and OsTom20, in particular between L158 of pSOD and W81 of OsTom20, as evidenced by analysis of intermolecular distances and corresponding relative free energy landscapes. L158 is part of an interacting LRTLA motif. These results provide new insight into the structural basis and dynamics of precursor recognition by Tom20 in plant, and their generality is supported by sequence alignments with seven other plants.

Mitochondrial Targeting of theArabidopsisF1-ATPase γ-Subunit via Multiple Compensatory and Synergistic Presequence Motifs

“…From these sequences, Tom20 was deduced to bind the consensus sequence fxxff (where f indicates a hydrophobic residue and x indicates any residue) (Okamoto et al, 2002;Obita et al, 2003;Rimmer et al, 2011;Yamamoto et al, 2011). In addition, NMR studies have demonstrated that Tom20 interacts with LRTLA and LRRFV in rice (Oryza sativa) SOD and Arabidopsis threonyl tRNA synthetase, respectively (Zhang et al, 2010). These plant motifs conform to the Tom20 consensus sequence derived from yeast PSs.…”

“…The result showing that pFAg contains only few motifs is rather surprising as many motifs are expected to participate in various steps of the protein import process. Previously, specific motifs were identified, including Tom20 binding sites and mtHsp70 binding sites (Okamoto et al, 2002;Obita et al, 2003;Zhang et al, 2010;Rimmer et al, 2011;Yamamoto et al, 2011). Moreover, the hydrophobic amino acid residues in the N-terminal region are important for efficient protein import into plant MT (Duby et al, 2001;Ambard-Bretteville et al, 2003;(Lee and Whelan, 2004).…”

“…Various biochemical approaches have been used to study the interaction of PSs with translocator complexes (Zhang et al, 2010;Rimmer et al, 2011;Saitoh et al, 2011). In this study, the localization of these mutants provided insight into the specific steps in which these motifs may be involved (Figure 9).…”

“…The configuration of positively charged and hydrophobic amino acids has been demonstrated to form an amphipathic a-helix in solution, and NMR analysis of the central mitochondrial outer membrane (MOM) import receptor, Tom20, in complex with an N-terminal PS revealed that the hydrophobic surface of the amphipathic a-helix binds to the hydrophobic groove of Tom20 (Abe et al, 2000). Recent studies revealed that Tom20 binding sites contain a consensus sequence of fxxff (where f indicates a hydrophobic residue and x indicates any amino acid residue) (Muto et al, 2001;Obita et al, 2003;Zhang et al, 2010;Yamamoto et al, 2011).…”

Isolation of Thylakoid Membrane Complexes from Rice by a New Double-Strips BN/SDS-PAGE and Bioinformatics Prediction of Stromal Ridge Subunits Interaction

“…The MM-GBSA approach show the advantage to estimate the binding free energy of the complex[30], [31]. In this study, the implicit generalized Born solvation model was used (igb = 2).…”