Modelling the free energy profile of the mitochondrial ADP/ATP carrier

Edmund R.S Kunji,Martin S King,Paul G Crichton,Roger Springett

doi:10.1016/j.bbabio.2017.05.006

Abstract

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.

Highlights

In the functioning mitochondrion the mitochondrial ADP/ATP carrier forms part of the mitochondrial ATP circuit, importing cytosolic ADP into the matrix and exporting ATP synthesized by the FoF1 ATP synthase into the intermembrane space, which is confluent with the cytosol [1], see [2] for a recent review
As with all mitochondrial carrier proteins, the ADP/ATP carrier consists of three homologous ~100 amino acid repeat domains [6,7], each composed of two transmembrane α-helices separated by a matrix loop and small α-helix [8]
We have shown that the stability of the carrier in detergent is independent of the cytosolic network strength when the carrier is locked in the cytoplasmic conformation with carboxyatractyloside, but is proportional to the network strength when the carrier is locked in the matrix conformation with bongkrekic acid, demonstrating that the cytoplasmic network is only interacting in the matrix conformation [21]

Summary

Introduction

In the functioning mitochondrion the mitochondrial ADP/ATP carrier forms part of the mitochondrial ATP circuit, importing cytosolic ADP into the matrix and exporting ATP synthesized by the FoF1 ATP synthase into the intermembrane space, which is confluent with the cytosol [1], see [2] for a recent review. As with all mitochondrial carrier proteins, the ADP/ATP carrier consists of three homologous ~100 amino acid repeat domains [6,7], each composed of two transmembrane α-helices separated by a matrix loop and small α-helix [8]. The atomic structures of the bovine and yeast ADP/ATP carriers inhibited by carboxyatractyloside show that the three repeat domains form a six α-helical barrel around a central cavity that is open to the intermembrane space [8,9]. By considering conservation of amino acids as well as distance and chemical constraints, and by analysis of the pseudo-symmetry of mitochondrial carriers, a single substrate-binding site was identified in the centre of the cavity [10,11,12]. ADP binds to this site in labelling studies [13] and molecular dynamics simulations [14,15,16]

Methods

Results

Conclusion