The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.
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