Identification of Cardiolipin Binding Sites on Membrane Proteins using an Accelerated Computational Membrane Model

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Cardiolipin (CDL), a negatively-charged lipid found primarily in bacterial and mitochondrial membranes, is believed to play a major role in membrane morphology, some hereditary heart diseases, as well as acting as a proton reservoir in the inner mitochondrial membrane. CDL has been shown to regulate many integral membrane proteins involved in bioenergetics, often binding with nanomolar affinity. In this study we incorporate CDL into the Highly Mobile Membrane Mimetic (HMMM), a membrane model consisting of lipid head groups partitioned by an organic solvent mimicking hydrophobic acyl tails, which allows for fast lipid diffusion, nanosecond spontaneous membrane insertion of proteins, and providing atomistic sampling of CDL-protein interactions, which gives rise to the identification of potential CDL binding residues. As an application, we chose CLC chloride transporter (CLC-ec1), an integral membrane protein involved in maintaining proton gradients, which initial experimental results show that CDL increases CLC-ec1 activity, likely due to CDLs ability to act as a proton reservoir. The increased lipid diffusion and sampling of CDL containing HMMM membranes aided in determining potential binding sites for ClC Chloride transporter. Incorporating CDL into the HMMM model involved applying harmonic restraints to each carbonyl carbon for each tail to maintain membrane thickness, as well as applying RMSD based collective variable restraints on ClC-ec1. Using ten one-hundred nanosecond HMMM simulations of CLC-ec1 embedded into a 80:20 POPE:CDL membrane, interactions between CDL and protein residues were measured. The overall microsecond simulation time, together with the enhanced dynamics offered by the HMMM membrane, provided a large sample size, which led to narrowing down the potential CDL binding sites to two sites, each stabilized by two basic side chains, thus expediting and guiding ClC-ec1 mutagenesis studies.