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

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ABSTRACT

The exchange of ADPand ATP across the inner mitochondrial membraneis a fundamental cellular process. This exchange is facilitated bythe adenine nucleotide translocase, the structure and function ofwhich are critically dependent on the signature phospholipid of mitochondria,cardiolipin (CL). Here we employ multiscale molecular dynamics simulationsto investigate CL interactions within a membrane environment. Usingsimulations at both coarse-grained and atomistic resolutions, we identifythree CL binding sites on the translocase, in agreement with thoseseen in crystal structures and inferred from nuclear magnetic resonancemeasurements. Characterization of the free energy landscape for laterallipid interaction via potential of mean force calculations demonstratesthe strength of interaction compared to those of binding sites onother mitochondrial membrane proteins, as well as their selectivityfor CL over other phospholipids. Extending the analysis to other membersof the family, yeast Aac2p and mouse uncoupling protein 2, suggestsa degree of conservation. Simulation of large patches of a model mitochondrialmembrane containing multiple copies of the translocase shows thatCL interactions persist in the presence of protein–proteininteractions and suggests CL may mediate interactions between translocases.This study provides a key example of how computational microscopymay be used to shed light on regulatory lipid–protein interactions.

No MeSH data available.


Organization of bovineANT1 in large membrane patches. (A) Finalsnapshot (t = 20 μs) of the matrix leafletshowing the organization of 25 ANT1 proteins in a PC/PE/CL mixed membrane.(B) Clustering dynamics shown as the percentage of ANT1 cluster sizeover the simulated time course, along with a snapshot of an ANT1 dimerwith CL mediating the interaction. Other lipid molecules have beenomitted for the sake of clarity. (C) Density plots showing the frequencyof occurrence of ANT around ANT, and CL around ANT. The protein hasbeen omitted from the CL plot for the sake of clarity.
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fig7: Organization of bovineANT1 in large membrane patches. (A) Finalsnapshot (t = 20 μs) of the matrix leafletshowing the organization of 25 ANT1 proteins in a PC/PE/CL mixed membrane.(B) Clustering dynamics shown as the percentage of ANT1 cluster sizeover the simulated time course, along with a snapshot of an ANT1 dimerwith CL mediating the interaction. Other lipid molecules have beenomitted for the sake of clarity. (C) Density plots showing the frequencyof occurrence of ANT around ANT, and CL around ANT. The protein hasbeen omitted from the CL plot for the sake of clarity.

Mentions: Examination of the final snapshot showeda degree of protein oligomerization (Figure 7A), with a mixture of monomers, dimers, andhigher-order oligomers observed. Analysis of the dynamics of proteinclustering showed the self-assembly of oligomers began on a submicrosecondtime scale (Figure 7B and Movie 2), with ∼50% of theproteins participating in an oligomeric state by 8 μs. Calculationof the averaged 2D protein density maps for ANT1 and CL showed thatthis interaction occurred at three specific interaction interfaces,which colocalized with the three CL binding sites identified previously(Figure 7C). Indeed,visual inspection of the simulations showed that CL molecules wereoften found at these interfaces, loosely bridging proteins. However,in contrast to the crystallographic dimers,29 we did not observe a stable arrangement in which two CL moleculeswere present between the two proteins. Such an arrangement may beunfavorable because of the proximity of the negatively charged (−2e) CL headgroups, as well as a degree of steric conflict.This is suggestive of possible competition between protein–proteinand protein–lipid interactions. Calculation of 2D density mapsand ANT-CL contacts for the first 1 μs of the simulation (duringwhich period the ANT molecules are predominantly monomeric) and thefinal 1 μs (during which period the ANT molecules are predominantlyoligomeric) revealed similar profiles, indicating the broad patternsof CL interaction remain similar under the two regimes (Figure S7). However, further simulations withimproved sampling and resolution would be needed to unpack any finerdetails of CL behavior within oligomers.


Lipid-Loving ANTs: Molecular Simulations of CardiolipinInteractions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes
Organization of bovineANT1 in large membrane patches. (A) Finalsnapshot (t = 20 μs) of the matrix leafletshowing the organization of 25 ANT1 proteins in a PC/PE/CL mixed membrane.(B) Clustering dynamics shown as the percentage of ANT1 cluster sizeover the simulated time course, along with a snapshot of an ANT1 dimerwith CL mediating the interaction. Other lipid molecules have beenomitted for the sake of clarity. (C) Density plots showing the frequencyof occurrence of ANT around ANT, and CL around ANT. The protein hasbeen omitted from the CL plot for the sake of clarity.
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Related In: Results  -  Collection

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fig7: Organization of bovineANT1 in large membrane patches. (A) Finalsnapshot (t = 20 μs) of the matrix leafletshowing the organization of 25 ANT1 proteins in a PC/PE/CL mixed membrane.(B) Clustering dynamics shown as the percentage of ANT1 cluster sizeover the simulated time course, along with a snapshot of an ANT1 dimerwith CL mediating the interaction. Other lipid molecules have beenomitted for the sake of clarity. (C) Density plots showing the frequencyof occurrence of ANT around ANT, and CL around ANT. The protein hasbeen omitted from the CL plot for the sake of clarity.
Mentions: Examination of the final snapshot showeda degree of protein oligomerization (Figure 7A), with a mixture of monomers, dimers, andhigher-order oligomers observed. Analysis of the dynamics of proteinclustering showed the self-assembly of oligomers began on a submicrosecondtime scale (Figure 7B and Movie 2), with ∼50% of theproteins participating in an oligomeric state by 8 μs. Calculationof the averaged 2D protein density maps for ANT1 and CL showed thatthis interaction occurred at three specific interaction interfaces,which colocalized with the three CL binding sites identified previously(Figure 7C). Indeed,visual inspection of the simulations showed that CL molecules wereoften found at these interfaces, loosely bridging proteins. However,in contrast to the crystallographic dimers,29 we did not observe a stable arrangement in which two CL moleculeswere present between the two proteins. Such an arrangement may beunfavorable because of the proximity of the negatively charged (−2e) CL headgroups, as well as a degree of steric conflict.This is suggestive of possible competition between protein–proteinand protein–lipid interactions. Calculation of 2D density mapsand ANT-CL contacts for the first 1 μs of the simulation (duringwhich period the ANT molecules are predominantly monomeric) and thefinal 1 μs (during which period the ANT molecules are predominantlyoligomeric) revealed similar profiles, indicating the broad patternsof CL interaction remain similar under the two regimes (Figure S7). However, further simulations withimproved sampling and resolution would be needed to unpack any finerdetails of CL behavior within oligomers.

View Article: PubMed Central - PubMed

ABSTRACT

The exchange of ADPand ATP across the inner mitochondrial membraneis a fundamental cellular process. This exchange is facilitated bythe adenine nucleotide translocase, the structure and function ofwhich are critically dependent on the signature phospholipid of mitochondria,cardiolipin (CL). Here we employ multiscale molecular dynamics simulationsto investigate CL interactions within a membrane environment. Usingsimulations at both coarse-grained and atomistic resolutions, we identifythree CL binding sites on the translocase, in agreement with thoseseen in crystal structures and inferred from nuclear magnetic resonancemeasurements. Characterization of the free energy landscape for laterallipid interaction via potential of mean force calculations demonstratesthe strength of interaction compared to those of binding sites onother mitochondrial membrane proteins, as well as their selectivityfor CL over other phospholipids. Extending the analysis to other membersof the family, yeast Aac2p and mouse uncoupling protein 2, suggestsa degree of conservation. Simulation of large patches of a model mitochondrialmembrane containing multiple copies of the translocase shows thatCL interactions persist in the presence of protein–proteininteractions and suggests CL may mediate interactions between translocases.This study provides a key example of how computational microscopymay be used to shed light on regulatory lipid–protein interactions.

No MeSH data available.