EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
Doi
Abstract
Mitochondrial ADP/ATP carriers provide key transport
steps in eukaryotic oxidative phosphorylation by importing
ADP into the mitochondrial matrix and by exporting syn-
thesized ATP to fuel cellular processes. Structures of the in-
hibited cytoplasmic- and matrix-open states have confirmed
an alternating access mechanism, but the molecular nature
of substrate binding is unresolved. Here, we investigate
all solvent-exposed residues in the translocation pathway in
both conformational states of the mitochondrial ADP/ATP
carrier. By combining binding studies with functional assays
using single alanine replacement variants, we identify a single substrate binding site in the center of the cavity. It consists
of three positively charged residues, which could bind the phosphate groups, and a cluster of three aliphatic and one aromatic residue, which could bind the adenosine moiety of M the nucleotides. In addition, two arginine/asparagine pairs
are identified, which are unique to ADP/ATP carriers but
play a role in binding. Importantly, the same residues are O involved in binding of both ADP and ATP, implying that
the import and export steps occur consecutively and in a N reversible way. The features of the identified binding site
explain the electrogenic nature of transport