The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.
A powerful
computational strategy to determine the equilibrium
association constant of two macromolecules with explicit-solvent molecular
dynamics (MD) simulations is the “geometric route”,
which considers the reversible physical separation of the bound complex
in solution. Nonetheless, multiple challenges remain to render this
type of methodology reliable and computationally efficient in practice.
In particular, in one, formulation of the geometric route relies on
the potential of mean force (PMF) for physically separating the two
binding partners restrained along a straight axis, which must be selected
prior to the calculation. However, practical applications indicate
that the calculation of the separation PMF along the predefined rectilinear
pathway may be suboptimal and slowly convergent. Recognizing that
a rectilinear straight separation pathway is generally not representative
of how the protein complex physically separates in solution, we put
forth a novel theoretical framework for binding free-energy calculations,
leaning on the optimal curvilinear minimum free-energy path (MFEP)
determined from the string method. The proposed formalism is validated
by comparing the results obtained using both rectilinear and curvilinear
pathways for a prototypical host–guest complex formed by cucurbit[7]uril
(CB[7]) binding benzene, and for the barnase–barstar protein
complex. On the basis of multi-microsecond MD calculations, we find
that the calculations following the traditional rectilinear pathway
and the string-based curvilinear pathway agree quantitatively, but
convergence is faster with the latter.
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