Abstract
The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.
Highlights
Membrane transporters are ubiquitous integral membrane proteins that mediate transfers of various substrates across the biological membrane
The present linear response path following (LRPF) molecular dynamics (MD) simulations for ~10 μs succeeded in predicting an atomic structure of the IF form through the accelerated simulation of the global conformational changes from the OF form to the IF one with root-mean-square deviation (RMSD) of ~9 Å (Fig 3A), which is unprecedentedly large as that obtained by the atomic-level MD simulations without a target structure
Even after the large conformational changes from the OF form, the predicted IF structure was found to be well-organized and very stable in MD simulations for 3–4 μs, which is completely different from expected behavior of unfolded proteins
Summary
Membrane transporters are ubiquitous integral membrane proteins that mediate transfers of various substrates across the biological membrane. Despite the diversity of transporters, their transport processes are often characterized by a simple model of the alternating access, where transport of the substrate is accomplished in a cycle of conformational transitions of the transporter protein between an outward-facing (OF) form and an inward-facing (IF) one. The large conformational changes of the transporter proteins essentially involved in the alternating. Atomistic modeling of alternating access of ADP/ATP carrier with molecular simulations. Industry and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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