Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes

Abstract

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions.

Insights into Membrane Protein-Lipid Interactions from Free Energy Calculations

“…They also represent three species of lipid ( Figure 1C) which frequently form interactions with membrane proteins, namely: PIP2, a negatively charged phospholipid which interacts with many ion channels and receptors in mammalian cell membranes; CDL, also negatively charged, which is present in mitochondrial and bacterial inner membranes, and cholesterol, which has been observed to bind to many G-protein coupled receptors (GPCRs) and ion channels. Thus, we probe the following interactions: of PIP2 with the mammalian inward rectifying potassium channel, Kir2.2; of CDL with the mitochondrial inner membrane ADP/ATP carrier protein, AAC 21 and with the bacterial inner membrane leucine transporter, LeuT 34,50 ;…”

“…Information on the structural basis of these interactions can be determined by X-ray crystallography, cryoelectron microscopy, and NMR. In contrast, determining the free energies of these interactions remains very challenging either experimentally 15,16,29 , or computationally 5,11,18,20,21 .…”

“…In this forcefield, molecules are coarse grained through the representation of approximately 4 heavy atoms and associated hydrogens as a single bead or particle. Whilst this simplification provides the force field with reduced resolution 27 , it has repeatedly been shown as highly proficient in the identification of specific interactions between proteins and membrane lipids, including cardiolipin (CDL) 11,21,28,29 , phosphatidylinositol (4,5) bisphosphate (PIP2) 5,14,20 , cholesterol 20,30 ( Figure 1C) and others.…”

“…Membrane proteins and lipids pose particular challenges of sampling and convergence for accurate free energy estimation 18 , arising from the relatively slow rates of lipid diffusion and from the diversity of lipid species present in complex biological membranes 19 . To date, most studies 5,11,18,20,21 have combined umbrella sampling with a potential of mean force (PMF) calculation along a one-dimensional reaction coordinate connecting the binding site with the surrounding membrane 18 ( Figure 1B). Convergence of such calculations (i.e.…”

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

“…Coarse-grained (CG) (Marrink et al, 2007) molecular dynamics simulations (Figure 1) enable the study of lipid interactions with integral membrane proteins (Hedger et al, 2016b). This approach provides access to the long simulation timescales and large system sizes required for thorough sampling of lipid-protein interaction space (Corradi et al, 2018).…”